ENVIRONMEN
I-AL RESEARCH
The Effect
14, 152 -163
(1977)
of Propoxur on Rats Fed Diets Differing Protein Content’ LIDIA
Received
in
PU~Y~~SKA
September
I?, 1976
In 6-week-old Wistar rats fed for 30 and 93 days either 4.5 or 26% casein diets containing 0, 500, 1500. 4500, and 9000 ppm of propoxur several biological changes were found which were more pronounced when propoxur was added to low-protein diet for 93 days, particularly in concentrations of 4500 and 9000 ppm. The changes were as follows. (I) In the liver-decreased activity of aromatic amino acids aminotransferases (AAA) and /3-glucuronidase (P-GR) as well as diminished protein content in supernatant: unchanged activity of aspartate aminotransferase (AspAT). glucosephosphate isomerase (PHI) and fructosediphosphate aldolase (ALD). (2) In the serumAecreased activity of acetylcholinesterase (AChE) as well as P-GR and increased PHI activity: unaltered activity of ALD and sorbitol dehydrogenase. (SDH). (3) In the brainAecreased activity of AChE, PHI. ALD. AAA. reduced protein content in supernatant: unchanged activity of AspAT and biogenic amines level. (4) Decreased rate of rat body weight gain on contaminated normoprotein diet and weight loss on contaminated low-protein diet. (S) Changes in the relative weight of certain internal organs: no detectable anatomical changes in the examined organs. The acute oral toxicity of propoxur was definitely enhanced by the previous feeding of low-protein diet: I .7 times in males and I .3 times in females after 30 days, and after 93 days 4.3 and 2.7 times, respectively.
INTRODUCTION
It is well known that many exogenous and endogenous factors exert an effect on the toxicity and metabolism of various substances penetrating into the organism, pesticides included (Parke and Williams, 1969; Remmer, 1970). The content and quality of protein in the diet belong to these exogenous factors. It has been demonstrated by many experiments that protein deficiency causes degenerative changes in the liver and delays its maturation, thus inhibiting synthesis of microsomal hepatic enzymes which participate in the metabolic processes of decomposition of foreign substances reaching the organism (McLean and McLean, 1969). McLean and McLean (1969) reported investigations demonstrating that dietary protein deficiency exerts different effects on the toxicity of various foreign chemicals in the organism. They claim that protein deficiency may augment, lessen. alter, or have no effect on the susceptibility of animals to the toxic action of various agents. In experiments on rats it has been demonstrated that, with dietary protein deficiency, the acute toxicity of orally administered pesticide is increased when it ’ This work was carried out under the financial support of the Plant Protection within the project 09.1.3. The grant from the World Health Organization used supplies is also gratefully acknowledged. 152 CopyrIght @ 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
Institute in Poznan for the purchase of
PROPOXUR
IN
RATS
153
is metabolized to less toxic metabolites, e.g., the toxicity of carbaryl increases 6 times and that of captan 26 times (Boyd, 1969: Boyd and Stefec, 1969). When the pesticide is metabolized to more toxic metabolites, the dietary protein deficiency impairing the metabolic processes attenuates its toxic effect on the organism. An example of this effect may be heptachlor. In this case the lowprotein diet results in lower formation of the four times as toxic epoxide (Weatherholtz et al., 1969). Dietary protein deficiency changes not only the acute toxicity of orally administered foreign substances, but affects also their long-term effect (Lee et al., 1964; Casterline and Williams, 1971). The quality of dietary protein is also important and it was observed, for example, that oral toxicity of phenacetin for rats was unaltered by diets containing optimal amounts of casein or lactalbumin, but was increased when soybean protein was used. In view of this, casein has been selected as a standard protein in toxicological investigations (Boyd et al.. 1969). The purpose of the present work was to investigate the effects of propoxur on rats fed diets differing in casein content. MATERIALS
AND METHODS
The investigations were carried out on male and female Wistar rats of the Food and Nutrition Institute colony, aged 6 weeks at the time of starting the experiments. The rats were kept during 30 and 93 days on a diet containing 4.5 or 26% casein with propoxur added to concentrations of 0,500, 1500,4500, and 9000 ppm. Each group comprised at least 80 animals, 40 of either sex, and each variant of experiment was repeated twice. The animals were kept in a room lighted during 12 hours (from 6 AM to 6 PM) and in darkness for another 12 hours. The diets containing 4.5 or 26% casein were prepared according to Hegsted and Chang (1965) and Boyd (1969) with the following modifications: sunflower oil was substituted for hydrogenated cottonseed oil and wheat starch for corn starch. Propoxur Bayer with 99.6% active substance obtained from “Azot” Chemical Laboratories in Jaworzno was added to the diets. Propoxur (Bayer 39007. Baygon, Unden) belongs to carbamate insecticides and is widely used in Poland as well as in the other countries. Its chemical name is 2-isopropoxyphenyl-N-methyl carbamate. All diets and drinking water were given ad lihitum. The food intake and weight gain were recorded daily. After 30 and after 93 days on these diets (in the case of diets containing propoxur 9000 ppm only after 93 days) the rats were killed by decapitation, always between 9 and 10 AM, and biological material was immediately taken for enzymatic and histological investigations. Enzyme activity measurements were performed in the 700-g supernatants from each brain and liver-tissue homogenates prepared in ice-cold physiological saline or glass-redistilled water. The methods used have been previously described in detail in the literature and are quoted in our recent publication (F’uiyfiska and Opuszynska-Freyer, 1975).
154
LIDIA
PUiYtiSKA
The tissue homogenates for enzymatic analyses are often, also in toxicological investigations, prepared in cold, distilled water or saline, for example, by Williams (1969), Casterline et al. (1971), Casterline and Williams (1971) or Pickering and Pickering (1971) for the estimation of the activity of /3-GR in the liver, cholinesterase and monoamine oxidase in the brain, cholinesterase and triacetinesterase in the brain and liver, or AChE in the brain, respectively. The results from enzymatic analyses presented here compare well with those obtained by the authors whose tissue homogenates were prepared in the same media as ours, as, for example, by Pickering and Pickering (1971) for the estimation of AChE activity in the brain or by Bernard and Ode11 (1950) for the liver P-GR activity determination. They are also consistent with those obtained by the authors, who used other homogenizing media as, for example, 0.25 M sucrose (Century, 1972), 0.14 M KC1 containing 0.0025 N NaOH (Hardeland, 1970), 0.2 M Tris-HCl (pH 8) containing 0.1 mM dithiothreitol (Scott et al., 1970), phosphate buffer (pH 7.4) (Fuller and Snoddy, 1968), 0.14 M KC1 (Wurtman er al., 1968; Wurtman and Larin, 1968), 0.14 M NaCl containing 0.02 M sodium phosphate (pH 7.6) (Singer and Mason, 1965, 1967), for the measuring of the liver tyrosine transaminase activity, or 0.32 M sucrose with EDTA and dithiothreitol (Gibb and Webb, 1969), 0.025 M sodium phosphate (pH 7.4) (Fuller, 1970), for the assay of the brain tyrosine transaminase activity, or 0.15 M KC1 for the estimation of the liver p-GR activity (Miettinen and Leskinen, 1963). After 93 days of feeding on both diets contaminated with 9000 ppm of propoxur the brain level of adrenaline and noradrenaline was determined by the method of Bertler et czl. (1958) and that of serotonin by the method of Bertler (1961) using Amberlite CG-50 in accordance with the description by Utley (1963): and the results were compared with the levels of these amines in the brains of rats fed for the same time control, noncontaminated diets. All tissue homogenates were prepared using a motor-driven Teflon-glass homogenizer. For histological examinations sections from the brain, heart, lungs, liver, kidneys, adrenals, pancreas, spleen, duodenum, small intestine, skeletal muscle, and testicles or ovaries were taken. The histological sections were evaluated by H. Molak-Olczakowa, M. B. from the Pathology Department, Institute of Biostructure, Medical Academy in Warsaw. The acute oral toxicity, LDso, of propoxur in rats after a 30- and 93-day lowprotein and normoprotein diet was estimated as well by the method of Weil(1952). The significance of the obtained differences between the values for control and propoxur-intoxicated groups has been calculated by the Student’s t test. The changes presented in the results were significant at least at P < 0.05. RESULTS The Effect of Propoxur on Body Weight, Weight of Internal as ++lell as Diet Intake in Rats
Organs
In concentrations of 4500 ppm and 9000 ppm, propoxur decreased the rate of weight gain in rats kept on the 26% casein diet. This was evident mainly during the
PROPOXUR
IN RATS
155
first days of the experiment, and could be explained by the lower intake of poisoned food in the first period of its administration. The weight gain was lower in comparison with rats kept on diets containing 0,500 or 1500 ppm of propoxur by 21%) in males and by 31% in females (Fig. la). Addition of 4500 or 9000 ppm of propoxur to the 4.5% casein diet caused evident weight loss in rats. After 93 days on this casein diet without propoxur or with only 500 or 1500 ppm of this pesticide, the mean weight loss was 4% of the initial weight in males and females. When propoxur concentration rose to 4500 ppm this weight loss increased to 18% in males and 27% in females. When the concentration of the pesticide was 9000 ppm the respective weight loss was 37 and 45% (Fig. la). Cholinergic disturbances were not observed at any of the used doses; in the groups receiving propoxur in the concentration of 9000 ppm only one rat died in each group. When propoxur concentrations in the diets were 4500 or 9000 ppm the food intake was lower only at the beginning of the experiment. Later, the food was ingested in amounts similar to those consumed by controls. The weight loss of rats receiving propoxur with low-protein diet, as observed throughout the experiment, suggests lower utilization of poisoned food. The effects of propoxur on the weight of internal organs were more evident when it was added to the low-protein diet, particularly for 93 days of the experiment. Significant differences were found in the weight of the liver, kidneys, brain, and testicles (Figs. lb, and 2a,b). An increase in the relative kidney and brain weight was mainly seen in rats fed for 93 days the 4.5% casein diet contaminated with 9000 ppm of propoxur (Fig. lb). Changes in testicular weight were also observed only after 93 days of feeding rats on low-protein contaminated by propoxur diet. When propoxur concentrations were 500, 1500, and 4500 ppm in that diet, the relative weight of testicles increased by 5 and 21%. respectively, but when the concentration was 9000 ppm, the testicular weight fell by 66% of the initial value (Fig. 2b). The relative weight of the liver was increased under the influence of propoxur in the same degree in rats kept on both contaminated diets (Fig. 2a). The Ef%ct of PU~~OXU~ OH the Activity of the Studied Enzynlcrtic Systems Serum AChE activity was inhibited in males to a greater degree when they were kept on the contaminated 4.5% casein diet. When males were fed the 26% casein diet no inhibition of this activity was observed after 30 days notwithstanding the propoxur concentration. After 93 days inhibition was noted at a concentration of 1500 ppm and more; but was still less than that after the same time in males receiving propoxur with the 4.5% casein diet (Fig. 2~). In females. on the other hand, inhibition of AChE activity in the serum appeared earlier when propoxur was given in the diet containing 26% of casein, but with increasing duration of the experiment it decreased, while in protein-deficient rats it increased. After 93 days at propoxur concentration of 9000 ppm AChE activity was inhibited in low-protein females by 72%, whereas in the normoprotein animals inhibition was only 37% of the control values (Fig. 2~).
156
LIDIA
PU%YhKA
Body 99
weight
change dd
0
’ BOOppm
26% cosem diet
@-30days
n
-93days
FIG. 1. The effect of increasing doses of propoxur administered with diets differing in protein content on (a) the body weight of rats. I, initial weight: II. weight on 5th day: III, on 11th day; IV. on 31st day: V, on 62nd day: VI, on 94th day of the experiment; (b) the weight of kidneys and brain in relation to the body weight of rats in experiments for determining the 30- and 93-day toxicity of propoxur.
In the brain of males as well as females the inhibition of AChE activity was of the same order after 30 days on contaminated 4.5 or 26% casein diets. However, after 93 days the degree of propoxur inhibition of AChE activity in the brain of protein-deficient rats at each concentration of the pesticide was higher than in the brain of normoprotein rats (Fig. 2d). In the brain the activity of enzymes participating in the carbohydrate metabolism, PHI and ALD, was decreased as well. Decreased PHI activity appeared after 93 days of low-protein diet contaminated by 4500 ppm of propoxur and it amounted to 17% of the control values in males as well as in females. This decrease became more pronounced when the dose amounted to 9000 ppm, reaching 30% of the control values. By this last dose of propoxur the activity was
PROPOXUR % body
IN RATS
157 Acetylchohnestera5e
weight
octivrty
Cl Liver
93
a Percent of Lontrol
YY
Serum Percent
of control
Brain
Fro. 2. The effect of increasing doses of propoxur administered with diets of different protein content on the weights of liver (a) and testicles(b) in relation to the body weight of rats; the rat serum(c) and brain (d) acetylcholinesterase activity expressed as a percentage of the control. Control values expressedin micromoles of acetylthiocholine hydrolyzed per minute per milliliter of serum or gram of wet brain are the following: on 4.5% protein diet and 26% protein diet after 30 days in females 0.46.0.89; in males 0.48. 0.38. and after 93 days 0.61. 0.84. 0.56. 0.51, respectively. Corresponding values for brain are 9.45. 11.39: 8.27. 12.63: 9.68. 9.28: 9.15. 7.66.
decreased also in rats kept on the 26% casein diet by about 20% of the control values (Fig. 3b). Similarly, the activity of ALD in the brain was lowest after 93 days on the diet with 9000 ppm of propoxur. In rats receiving this concentration of this pesticide in the 4.5% casein diet activity was decreased by about 25% of the control values. In males this activity fell from 359 ? 8.4 to 279.2 f 16.8 and in females from 359.8 ? 8.4 to 263 5 14.1 mm3 of fructose- 1,6-diphosphate decomposed in 1 minute by 1 g of wet brain tissue. In rats receiving this concentration of propoxur with 26% casein diet the activity was 6% lower, on the average, than in controls (difference statistically not significant). In males this activity dropped from 333.8 5 18.5 to 308.9 + 10.5 and in females from 335.7 + 13.7 to 317.9 ‘-c 15.2 mm3 of fructose-l, 6-diphosphate decomposed in 1 minute by 1 g of wet brain tissue. At each mean value of activity the standard deviation is given. When propoxur was added to the diet in the brain of rats, decreased activity of AAA was also noted (Fig. 4). The phenylalanine aminotransferase activity dropped to lower values when propoxur was added to the 4.5% casein diet and this activity fall was after 30 days of the same order as after 93 days.
158
LIDIA
PUiYhKA
Serum
Serum Activiiq
a -4,5Xco& {I diet
Activity
Q-2BLco5Gn
diet
3. Comparison of the activity of (a) glucosephosphate isomerase in the serum. (b) glucosephosphate isomerase in the brain, (c) P-glucuronidase in the serum. td) P-glucuronidase in the liver, of rats receiving increasing doses of propoxur in diets with different protein levels. The activity of glucosephosphate isomerase is expressed in pmoles of fructose-6-phosphate formed per minute per milliliter of serum or gram of wet brain. The activity ofp-glucuronidase is expressed in nanomoles ofphenolphtalein formed per minute per milliliter of serum or gram of wet liver. FIG.
In the case of tyrosine and tryptophan aminotransferase the activity was more reduced when propoxur was given with the 26% casein diet but just after 93 days. In the case of tryptophan aminotransferase an increase in the activity in the brain was observed after 30 days with propoxur concentrations of 1500 and 4500 ppm in the 4.5% casein diet. No significant differences were observed in the level of biogenic amines in the brain of rats after 93 days on the diet contaminated by 9000 ppm of propoxur without regard to the casein concentration. A statistically significant decrease of the activity of AAA was found also in the liver; this decrease was more pronounced in rats kept on contaminated proteindeficient diet (Fig. 4). After 93 days on low-protein diet with 9000 ppm of propoxur a slight, although statistically significant, fall in the protein content was noted in the brain and liver supernatants. This fall was of similar order in males and females. In the brain this mean fall was 15%. and in the liver 19% of the control values. No changes were observed in the activity of ALD and SDH in the serum at any concentrations of propoxur added irrespective of diet fed. The activity of ALD and that of AspAT were unchanged in the liver and that of AspAT also in the brain. The activity of serum PHI increased with the concentrations of propoxur in the
PROPOXLIR
4,YLcasein
26%case1n
diet
IN
diet
Brain
159
RATS
4,59msein db
AGIIVI~~
26kcoseln
diet 8
Liver
dd a
,oActi;lty~o ”
diet ?? 20
0 KQ ppm 1500 ppm 1500 ppm 3000 ppm 0 5CQ wm ‘500 ppm 1500 ppm m0Pm 0 XXI ppm 5% wm 5DOppm )mPPm
q Xdoy,
l - 93duy5
FIG. 4. Transmination activity towards aromatic amino acids: t.-phenylalanine (a). r.-tyrosine (b), and r.-tryptophan (c) in the brain and liver of rats receiving diets with different protein content contaminated by increasing doses of propoxur. The activity is expressed in nanomoles of aromatic u-oxoacid formed per minute per gram of wet tissue.
diet and this activity increase was slightly greater in rats kept on low-protein diet but more marked after 93 days of the experiment duration (Fig. 3a). This increase was not accompanied by any changes of this enzyme activity in the liver. From among the applied concentrations of propoxur only 9000 ppm caused the decrease, by over 30% in relation to controls, of P-GR activity in the serum. This decrease was noted after 93 days of feeding rats on a 4.5% casein diet contaminated by the said dose of propoxur (Fig. 3~). The activity of this enzyme was decreased significantly also in the liver after 93 days and this decrease paralleled the increasing concentrations of propoxur in the 4.5% casein as well as in the 26% casein diet (Fig. 3d). Results
of Histological
Examinations
of Organs
In the present experiments propoxur was without any significant effects on the structure of the studied organs of rats, both, when administered with diets during 30 or 93 days or when given intragastrically for determination of the oral LD5,. Acute Oral Toxicity
of Proposer
The acute oral toxicity of propoxur expressed as LD,, in milligrams per kilogram of body weight was after 30 days of low-protein or normoprotein diet 67.1 ? 12.6 and 116.7 ? 15.4, respectively, in males and 73.8 -+ 9.8 and 102.4 ? 15.3 in females. After 93 days these values were 31.0 t 4.1; 133.6 t 17.8; 36.7 ? 3.9; 98.3 2 21.2, respectively. The above values of LD,,, are arithmetical means (kstandard
160 deviation) each.
LIDIA
of at least five determinations
PUiYfiSKA
done in seven groups of at least six rats
DISCUSSION
It may be affirmed, in the light of the already described investigations, that certain results indicate a higher susceptibility of rats to propoxur given in lowprotein diet as compared with administration in normoprotein diet. It was demonstrated in these investigations that propoxur given in low-protein diet had a much greater inhibitory effect on the weight gain of rats, especially females, than when it was given in normoprotein diet. The relative weight of brain and kidneys was significantly more increased when propoxur was given in low-protein diet. As regards the investigations of Feron et al. (1973), the increase in the relative weight of brain found in our rats on low-protein diet contaminated by the highest concentrations of propoxur may be explained mainly by the considerable loss of rat body weight on these diets, and not by the direct action of this pesticide on this organ. But the increase in the relative weight of kidney (and that of liver) seen in our experiments may be regarded as the result of the direct action of propoxur. Feron et al. (1973) found that when the rate of weight gain was reduced, the relative testicular weight decreased after the first 4 weeks of the experiment and increased after 13 weeks. In our investigations the significant rise in the relative weight of testicles after 93 days with propoxur concentration of 4500 ppm in the low-protein diet may be connected with the reduced gain body weight rate at this concentration of the pesticide, while the sudden fall of the relative testicular weight at 9000 ppm of propoxur with the same kind of diet was probably due to the direct toxic effect of the pesticide on this organ. Changes in the activity of the tested enzymes were similarly more pronounced when the animals received propoxur in the low-protein diet. These changes, although statistically significant, were small and their appearance at relatively high concentrations of propoxur (mostly at 4500 and 9000 ppm) indicates that they may be a threat to the organism, especially with a protein-deficient diet, only when high concentrations of propoxur are ingested during a long time period. The increased sensitivity of rats kept on diet insufficient in protein content to the toxic action of propoxur was more apparent when the preparation was given in a single dose through a gastric tube into the stomach than during long-term administration in diet. This sensitivity increased with longer administration of lowprotein diet. After 30 days the oral LD,, determined in rats on a low-protein diet was lower than in those kept on normoprotein diet 1.7 times in males and 1.3 times in females. After 93 days the LD,,, was lower, 4.3 times in males and 2.7 times in females, on a low-protein diet than in those on a normoprotein diet. In a paper published in 1969 Boyd and Stefec reported that the acute oral toxicity of 11 out of 12 pesticides studied in their laboratory increased two to eight times in rats kept for 28 days from weaning on a diet containing 3.5% of casein, as compared with rats kept on a diet with 26% of casein. The authors thought that this toxicity rise was not so important as to require investigations on humans for
PROPOXUR
IN
RATS
I61
establishing whether the use of these pesticides should be banned or limited in countries where the diet of the population is deficient in protein. It appears that this is also the case as regards the presently studied propoxur. This pesticide caused undesirable effects in rats when it was added to the diets chiefly in the highest concentrations and particularly after longer duration of the experiment. But it seems that propoxur, if properly handled in practice, should not penetrate into the organism in amounts capable of causing a harmful effect, especially if the diet of the population is, in general, deficient in protein. Finally, it is worth adding that we could not demonstrate a correlation between the level of biogenic amines and the activity of aromatic amino acids aminotransferases in the brain, although Gibb and Webb (1969) discussed the possibility that low activity of tyrosine aminotransferase in the brain suggests a fall in the level of biogenic amines there. In addition, it may be said that the decrease in the activity of aromatic amino acids aminotransferases in rat liver could be due to changes in the rhythm of food intake caused by propoxur or in the utilization of the diets given during the experiments. These factors could have caused alterations in the hepatic amino acid pool which regulates the activity of these enzymes (Rosen and Milholland, 1968; Fuller and Snoddy, 1968; Wurtman et al., 1968; Zigmond et al., 1969). The significantly lowered tyrosine transaminase activity was found by Century i 1972) in liver preparations from rats fed linseed or menhaden-oil-containing diets in comparison with animals fed low-polyunsaturated-fatty-acid-containing diets. The author stated that the significance of these results was not clear at that time and he did not suggest any probable mechanism for this relationship. Differences in the effects of propoxur on the transamination activity towards particular aromatic amino acids in the brain as well as in the liver support the view that different enzymes are responsible for transamination of these amino acids (Fonnum et al., 1964; Puiynska, 1972). REFERENCES Bernard, R. M., and Odell, L. D. (1950). Studies on beta-glucuronidase activity in pregnant albino rats. J. Luh. Clin. Med. 35, 940-944. Bertler. A. (1961). Effect of reserpine on the storage of catechol amines in brain and other tissues. Acra Physiol. Stand. St, 75-83. Bertler, A.. Carlsson, A.. and Rosengren. E. (1958). A method for the fluorimetric determination of adrenaline and noradrenaline in tissues. Aua Pllysiol. Scmzd. 44, 273-292. Boyd, E. M. (1961). Dietary protein and pesticide toxicity in male weanling rats. Bull. W. H. 0. 40, 801-805. Boyd, E. M.. Boulanger. M. A.. and De Castro, E. S. (1969). Phenacetin toxicity and dietary protein. Phurmacol. Res. Commun. 1, 15-19. Boyd, E. M., and Stefec, J. (1969). Dietary protein and pesticide toxicity. Cnud. Med. As.~oc~. J. 101, 33X-339. Cab Sci. 9, Part I]. l133- 1141. Singer, S., and Mason. M. (1965). Tyrosine-a-ketoglutarate transaminase. effect of the administration of sodium benzoate and related compounds on the hepatic enzyme level. Biochim. Biophys. Actrr 110, 370-379. Singer. S.. and Mason. M. (1967). The effects of the administration of sodium benzoate and diethylstilbestrol disulfate on the hepatic levels of several glucocorticoid-sensitive enzymes in adrenalectomired rats. &o&m. Birqhys. Acto. 146, 443-45 1. Singer. S.. and Mason. M. (1967). Studies of the in vitro and in vivo effects of conjugated steroids and carboxylic acids on hepatic tyrosine transaminase in the rat. Bioc.hirn. Biophvs. Acta 146, 451-466. Utley. J. D. (1963). The effects of anthranilic hydroxamic acid on rat behaviour and rat brain y-aminobutyric acid. norepinephrine and S-hydroxytryptamine concentrations. .I. Neurockm. 10, 423-431. Weatherholtz, W. M., Campbell, T. C.. and Webb, R. E. (1969). Effect of dietary protein levels on the toxicity and metabolism of heptachlor. J. N~itr. 98, 90-94. Wei]. C. S. (1953). Tables for convenient calculation of median-effective dose. LD,,, or ED,,,. and instructions in their use. Biomcrric,s 8, 249-163. Williams. C. H. (1969). p-glucuronidase activity in the serum and liver of rats administered pesticides and hepatotoxic agents. To.uiw[. Appl. Piwrmac~oi. 14, -783-29-7.
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IN
RATS
163
Wurtman. R. J., and Larin, F. (1968). A sensitive and specific isotopic assay for the estimation of tyrosine transaminase. Biochrn7. Pharmc7c~ol. 17, 817-818. Wurtman, R. J., Shoemaker. W. J.. and Larin. F. (1968). Mechanism of the daily rhythm in hepatic tyrosine transaminase activity: Role of dietary tryptophan. hoc. Nut. Acod. Sci. U.S.A. 59, 800-897. Zigmond, M. J.. Shoemaker. W. J., Larin. F.. and Wurtman, R. J. (1969). Hepatic tyrosine transaminase rhythm: Interaction of environmental lighting, food consumption and dietary protein content. .I. Ntrtr. 98. 71-75.
I-AL RESEARCH
The Effect
14, 152 -163
(1977)
of Propoxur on Rats Fed Diets Differing Protein Content’ LIDIA
Received
in
PU~Y~~SKA
September
I?, 1976
In 6-week-old Wistar rats fed for 30 and 93 days either 4.5 or 26% casein diets containing 0, 500, 1500. 4500, and 9000 ppm of propoxur several biological changes were found which were more pronounced when propoxur was added to low-protein diet for 93 days, particularly in concentrations of 4500 and 9000 ppm. The changes were as follows. (I) In the liver-decreased activity of aromatic amino acids aminotransferases (AAA) and /3-glucuronidase (P-GR) as well as diminished protein content in supernatant: unchanged activity of aspartate aminotransferase (AspAT). glucosephosphate isomerase (PHI) and fructosediphosphate aldolase (ALD). (2) In the serumAecreased activity of acetylcholinesterase (AChE) as well as P-GR and increased PHI activity: unaltered activity of ALD and sorbitol dehydrogenase. (SDH). (3) In the brainAecreased activity of AChE, PHI. ALD. AAA. reduced protein content in supernatant: unchanged activity of AspAT and biogenic amines level. (4) Decreased rate of rat body weight gain on contaminated normoprotein diet and weight loss on contaminated low-protein diet. (S) Changes in the relative weight of certain internal organs: no detectable anatomical changes in the examined organs. The acute oral toxicity of propoxur was definitely enhanced by the previous feeding of low-protein diet: I .7 times in males and I .3 times in females after 30 days, and after 93 days 4.3 and 2.7 times, respectively.
INTRODUCTION
It is well known that many exogenous and endogenous factors exert an effect on the toxicity and metabolism of various substances penetrating into the organism, pesticides included (Parke and Williams, 1969; Remmer, 1970). The content and quality of protein in the diet belong to these exogenous factors. It has been demonstrated by many experiments that protein deficiency causes degenerative changes in the liver and delays its maturation, thus inhibiting synthesis of microsomal hepatic enzymes which participate in the metabolic processes of decomposition of foreign substances reaching the organism (McLean and McLean, 1969). McLean and McLean (1969) reported investigations demonstrating that dietary protein deficiency exerts different effects on the toxicity of various foreign chemicals in the organism. They claim that protein deficiency may augment, lessen. alter, or have no effect on the susceptibility of animals to the toxic action of various agents. In experiments on rats it has been demonstrated that, with dietary protein deficiency, the acute toxicity of orally administered pesticide is increased when it ’ This work was carried out under the financial support of the Plant Protection within the project 09.1.3. The grant from the World Health Organization used supplies is also gratefully acknowledged. 152 CopyrIght @ 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
Institute in Poznan for the purchase of
PROPOXUR
IN
RATS
153
is metabolized to less toxic metabolites, e.g., the toxicity of carbaryl increases 6 times and that of captan 26 times (Boyd, 1969: Boyd and Stefec, 1969). When the pesticide is metabolized to more toxic metabolites, the dietary protein deficiency impairing the metabolic processes attenuates its toxic effect on the organism. An example of this effect may be heptachlor. In this case the lowprotein diet results in lower formation of the four times as toxic epoxide (Weatherholtz et al., 1969). Dietary protein deficiency changes not only the acute toxicity of orally administered foreign substances, but affects also their long-term effect (Lee et al., 1964; Casterline and Williams, 1971). The quality of dietary protein is also important and it was observed, for example, that oral toxicity of phenacetin for rats was unaltered by diets containing optimal amounts of casein or lactalbumin, but was increased when soybean protein was used. In view of this, casein has been selected as a standard protein in toxicological investigations (Boyd et al.. 1969). The purpose of the present work was to investigate the effects of propoxur on rats fed diets differing in casein content. MATERIALS
AND METHODS
The investigations were carried out on male and female Wistar rats of the Food and Nutrition Institute colony, aged 6 weeks at the time of starting the experiments. The rats were kept during 30 and 93 days on a diet containing 4.5 or 26% casein with propoxur added to concentrations of 0,500, 1500,4500, and 9000 ppm. Each group comprised at least 80 animals, 40 of either sex, and each variant of experiment was repeated twice. The animals were kept in a room lighted during 12 hours (from 6 AM to 6 PM) and in darkness for another 12 hours. The diets containing 4.5 or 26% casein were prepared according to Hegsted and Chang (1965) and Boyd (1969) with the following modifications: sunflower oil was substituted for hydrogenated cottonseed oil and wheat starch for corn starch. Propoxur Bayer with 99.6% active substance obtained from “Azot” Chemical Laboratories in Jaworzno was added to the diets. Propoxur (Bayer 39007. Baygon, Unden) belongs to carbamate insecticides and is widely used in Poland as well as in the other countries. Its chemical name is 2-isopropoxyphenyl-N-methyl carbamate. All diets and drinking water were given ad lihitum. The food intake and weight gain were recorded daily. After 30 and after 93 days on these diets (in the case of diets containing propoxur 9000 ppm only after 93 days) the rats were killed by decapitation, always between 9 and 10 AM, and biological material was immediately taken for enzymatic and histological investigations. Enzyme activity measurements were performed in the 700-g supernatants from each brain and liver-tissue homogenates prepared in ice-cold physiological saline or glass-redistilled water. The methods used have been previously described in detail in the literature and are quoted in our recent publication (F’uiyfiska and Opuszynska-Freyer, 1975).
154
LIDIA
PUiYtiSKA
The tissue homogenates for enzymatic analyses are often, also in toxicological investigations, prepared in cold, distilled water or saline, for example, by Williams (1969), Casterline et al. (1971), Casterline and Williams (1971) or Pickering and Pickering (1971) for the estimation of the activity of /3-GR in the liver, cholinesterase and monoamine oxidase in the brain, cholinesterase and triacetinesterase in the brain and liver, or AChE in the brain, respectively. The results from enzymatic analyses presented here compare well with those obtained by the authors whose tissue homogenates were prepared in the same media as ours, as, for example, by Pickering and Pickering (1971) for the estimation of AChE activity in the brain or by Bernard and Ode11 (1950) for the liver P-GR activity determination. They are also consistent with those obtained by the authors, who used other homogenizing media as, for example, 0.25 M sucrose (Century, 1972), 0.14 M KC1 containing 0.0025 N NaOH (Hardeland, 1970), 0.2 M Tris-HCl (pH 8) containing 0.1 mM dithiothreitol (Scott et al., 1970), phosphate buffer (pH 7.4) (Fuller and Snoddy, 1968), 0.14 M KC1 (Wurtman er al., 1968; Wurtman and Larin, 1968), 0.14 M NaCl containing 0.02 M sodium phosphate (pH 7.6) (Singer and Mason, 1965, 1967), for the measuring of the liver tyrosine transaminase activity, or 0.32 M sucrose with EDTA and dithiothreitol (Gibb and Webb, 1969), 0.025 M sodium phosphate (pH 7.4) (Fuller, 1970), for the assay of the brain tyrosine transaminase activity, or 0.15 M KC1 for the estimation of the liver p-GR activity (Miettinen and Leskinen, 1963). After 93 days of feeding on both diets contaminated with 9000 ppm of propoxur the brain level of adrenaline and noradrenaline was determined by the method of Bertler et czl. (1958) and that of serotonin by the method of Bertler (1961) using Amberlite CG-50 in accordance with the description by Utley (1963): and the results were compared with the levels of these amines in the brains of rats fed for the same time control, noncontaminated diets. All tissue homogenates were prepared using a motor-driven Teflon-glass homogenizer. For histological examinations sections from the brain, heart, lungs, liver, kidneys, adrenals, pancreas, spleen, duodenum, small intestine, skeletal muscle, and testicles or ovaries were taken. The histological sections were evaluated by H. Molak-Olczakowa, M. B. from the Pathology Department, Institute of Biostructure, Medical Academy in Warsaw. The acute oral toxicity, LDso, of propoxur in rats after a 30- and 93-day lowprotein and normoprotein diet was estimated as well by the method of Weil(1952). The significance of the obtained differences between the values for control and propoxur-intoxicated groups has been calculated by the Student’s t test. The changes presented in the results were significant at least at P < 0.05. RESULTS The Effect of Propoxur on Body Weight, Weight of Internal as ++lell as Diet Intake in Rats
Organs
In concentrations of 4500 ppm and 9000 ppm, propoxur decreased the rate of weight gain in rats kept on the 26% casein diet. This was evident mainly during the
PROPOXUR
IN RATS
155
first days of the experiment, and could be explained by the lower intake of poisoned food in the first period of its administration. The weight gain was lower in comparison with rats kept on diets containing 0,500 or 1500 ppm of propoxur by 21%) in males and by 31% in females (Fig. la). Addition of 4500 or 9000 ppm of propoxur to the 4.5% casein diet caused evident weight loss in rats. After 93 days on this casein diet without propoxur or with only 500 or 1500 ppm of this pesticide, the mean weight loss was 4% of the initial weight in males and females. When propoxur concentration rose to 4500 ppm this weight loss increased to 18% in males and 27% in females. When the concentration of the pesticide was 9000 ppm the respective weight loss was 37 and 45% (Fig. la). Cholinergic disturbances were not observed at any of the used doses; in the groups receiving propoxur in the concentration of 9000 ppm only one rat died in each group. When propoxur concentrations in the diets were 4500 or 9000 ppm the food intake was lower only at the beginning of the experiment. Later, the food was ingested in amounts similar to those consumed by controls. The weight loss of rats receiving propoxur with low-protein diet, as observed throughout the experiment, suggests lower utilization of poisoned food. The effects of propoxur on the weight of internal organs were more evident when it was added to the low-protein diet, particularly for 93 days of the experiment. Significant differences were found in the weight of the liver, kidneys, brain, and testicles (Figs. lb, and 2a,b). An increase in the relative kidney and brain weight was mainly seen in rats fed for 93 days the 4.5% casein diet contaminated with 9000 ppm of propoxur (Fig. lb). Changes in testicular weight were also observed only after 93 days of feeding rats on low-protein contaminated by propoxur diet. When propoxur concentrations were 500, 1500, and 4500 ppm in that diet, the relative weight of testicles increased by 5 and 21%. respectively, but when the concentration was 9000 ppm, the testicular weight fell by 66% of the initial value (Fig. 2b). The relative weight of the liver was increased under the influence of propoxur in the same degree in rats kept on both contaminated diets (Fig. 2a). The Ef%ct of PU~~OXU~ OH the Activity of the Studied Enzynlcrtic Systems Serum AChE activity was inhibited in males to a greater degree when they were kept on the contaminated 4.5% casein diet. When males were fed the 26% casein diet no inhibition of this activity was observed after 30 days notwithstanding the propoxur concentration. After 93 days inhibition was noted at a concentration of 1500 ppm and more; but was still less than that after the same time in males receiving propoxur with the 4.5% casein diet (Fig. 2~). In females. on the other hand, inhibition of AChE activity in the serum appeared earlier when propoxur was given in the diet containing 26% of casein, but with increasing duration of the experiment it decreased, while in protein-deficient rats it increased. After 93 days at propoxur concentration of 9000 ppm AChE activity was inhibited in low-protein females by 72%, whereas in the normoprotein animals inhibition was only 37% of the control values (Fig. 2~).
156
LIDIA
PU%YhKA
Body 99
weight
change dd
0
’ BOOppm
26% cosem diet
@-30days
n
-93days
FIG. 1. The effect of increasing doses of propoxur administered with diets differing in protein content on (a) the body weight of rats. I, initial weight: II. weight on 5th day: III, on 11th day; IV. on 31st day: V, on 62nd day: VI, on 94th day of the experiment; (b) the weight of kidneys and brain in relation to the body weight of rats in experiments for determining the 30- and 93-day toxicity of propoxur.
In the brain of males as well as females the inhibition of AChE activity was of the same order after 30 days on contaminated 4.5 or 26% casein diets. However, after 93 days the degree of propoxur inhibition of AChE activity in the brain of protein-deficient rats at each concentration of the pesticide was higher than in the brain of normoprotein rats (Fig. 2d). In the brain the activity of enzymes participating in the carbohydrate metabolism, PHI and ALD, was decreased as well. Decreased PHI activity appeared after 93 days of low-protein diet contaminated by 4500 ppm of propoxur and it amounted to 17% of the control values in males as well as in females. This decrease became more pronounced when the dose amounted to 9000 ppm, reaching 30% of the control values. By this last dose of propoxur the activity was
PROPOXUR % body
IN RATS
157 Acetylchohnestera5e
weight
octivrty
Cl Liver
93
a Percent of Lontrol
YY
Serum Percent
of control
Brain
Fro. 2. The effect of increasing doses of propoxur administered with diets of different protein content on the weights of liver (a) and testicles(b) in relation to the body weight of rats; the rat serum(c) and brain (d) acetylcholinesterase activity expressed as a percentage of the control. Control values expressedin micromoles of acetylthiocholine hydrolyzed per minute per milliliter of serum or gram of wet brain are the following: on 4.5% protein diet and 26% protein diet after 30 days in females 0.46.0.89; in males 0.48. 0.38. and after 93 days 0.61. 0.84. 0.56. 0.51, respectively. Corresponding values for brain are 9.45. 11.39: 8.27. 12.63: 9.68. 9.28: 9.15. 7.66.
decreased also in rats kept on the 26% casein diet by about 20% of the control values (Fig. 3b). Similarly, the activity of ALD in the brain was lowest after 93 days on the diet with 9000 ppm of propoxur. In rats receiving this concentration of this pesticide in the 4.5% casein diet activity was decreased by about 25% of the control values. In males this activity fell from 359 ? 8.4 to 279.2 f 16.8 and in females from 359.8 ? 8.4 to 263 5 14.1 mm3 of fructose- 1,6-diphosphate decomposed in 1 minute by 1 g of wet brain tissue. In rats receiving this concentration of propoxur with 26% casein diet the activity was 6% lower, on the average, than in controls (difference statistically not significant). In males this activity dropped from 333.8 5 18.5 to 308.9 + 10.5 and in females from 335.7 + 13.7 to 317.9 ‘-c 15.2 mm3 of fructose-l, 6-diphosphate decomposed in 1 minute by 1 g of wet brain tissue. At each mean value of activity the standard deviation is given. When propoxur was added to the diet in the brain of rats, decreased activity of AAA was also noted (Fig. 4). The phenylalanine aminotransferase activity dropped to lower values when propoxur was added to the 4.5% casein diet and this activity fall was after 30 days of the same order as after 93 days.
158
LIDIA
PUiYhKA
Serum
Serum Activiiq
a -4,5Xco& {I diet
Activity
Q-2BLco5Gn
diet
3. Comparison of the activity of (a) glucosephosphate isomerase in the serum. (b) glucosephosphate isomerase in the brain, (c) P-glucuronidase in the serum. td) P-glucuronidase in the liver, of rats receiving increasing doses of propoxur in diets with different protein levels. The activity of glucosephosphate isomerase is expressed in pmoles of fructose-6-phosphate formed per minute per milliliter of serum or gram of wet brain. The activity ofp-glucuronidase is expressed in nanomoles ofphenolphtalein formed per minute per milliliter of serum or gram of wet liver. FIG.
In the case of tyrosine and tryptophan aminotransferase the activity was more reduced when propoxur was given with the 26% casein diet but just after 93 days. In the case of tryptophan aminotransferase an increase in the activity in the brain was observed after 30 days with propoxur concentrations of 1500 and 4500 ppm in the 4.5% casein diet. No significant differences were observed in the level of biogenic amines in the brain of rats after 93 days on the diet contaminated by 9000 ppm of propoxur without regard to the casein concentration. A statistically significant decrease of the activity of AAA was found also in the liver; this decrease was more pronounced in rats kept on contaminated proteindeficient diet (Fig. 4). After 93 days on low-protein diet with 9000 ppm of propoxur a slight, although statistically significant, fall in the protein content was noted in the brain and liver supernatants. This fall was of similar order in males and females. In the brain this mean fall was 15%. and in the liver 19% of the control values. No changes were observed in the activity of ALD and SDH in the serum at any concentrations of propoxur added irrespective of diet fed. The activity of ALD and that of AspAT were unchanged in the liver and that of AspAT also in the brain. The activity of serum PHI increased with the concentrations of propoxur in the
PROPOXLIR
4,YLcasein
26%case1n
diet
IN
diet
Brain
159
RATS
4,59msein db
AGIIVI~~
26kcoseln
diet 8
Liver
dd a
,oActi;lty~o ”
diet ?? 20
0 KQ ppm 1500 ppm 1500 ppm 3000 ppm 0 5CQ wm ‘500 ppm 1500 ppm m0Pm 0 XXI ppm 5% wm 5DOppm )mPPm
q Xdoy,
l - 93duy5
FIG. 4. Transmination activity towards aromatic amino acids: t.-phenylalanine (a). r.-tyrosine (b), and r.-tryptophan (c) in the brain and liver of rats receiving diets with different protein content contaminated by increasing doses of propoxur. The activity is expressed in nanomoles of aromatic u-oxoacid formed per minute per gram of wet tissue.
diet and this activity increase was slightly greater in rats kept on low-protein diet but more marked after 93 days of the experiment duration (Fig. 3a). This increase was not accompanied by any changes of this enzyme activity in the liver. From among the applied concentrations of propoxur only 9000 ppm caused the decrease, by over 30% in relation to controls, of P-GR activity in the serum. This decrease was noted after 93 days of feeding rats on a 4.5% casein diet contaminated by the said dose of propoxur (Fig. 3~). The activity of this enzyme was decreased significantly also in the liver after 93 days and this decrease paralleled the increasing concentrations of propoxur in the 4.5% casein as well as in the 26% casein diet (Fig. 3d). Results
of Histological
Examinations
of Organs
In the present experiments propoxur was without any significant effects on the structure of the studied organs of rats, both, when administered with diets during 30 or 93 days or when given intragastrically for determination of the oral LD5,. Acute Oral Toxicity
of Proposer
The acute oral toxicity of propoxur expressed as LD,, in milligrams per kilogram of body weight was after 30 days of low-protein or normoprotein diet 67.1 ? 12.6 and 116.7 ? 15.4, respectively, in males and 73.8 -+ 9.8 and 102.4 ? 15.3 in females. After 93 days these values were 31.0 t 4.1; 133.6 t 17.8; 36.7 ? 3.9; 98.3 2 21.2, respectively. The above values of LD,,, are arithmetical means (kstandard
160 deviation) each.
LIDIA
of at least five determinations
PUiYfiSKA
done in seven groups of at least six rats
DISCUSSION
It may be affirmed, in the light of the already described investigations, that certain results indicate a higher susceptibility of rats to propoxur given in lowprotein diet as compared with administration in normoprotein diet. It was demonstrated in these investigations that propoxur given in low-protein diet had a much greater inhibitory effect on the weight gain of rats, especially females, than when it was given in normoprotein diet. The relative weight of brain and kidneys was significantly more increased when propoxur was given in low-protein diet. As regards the investigations of Feron et al. (1973), the increase in the relative weight of brain found in our rats on low-protein diet contaminated by the highest concentrations of propoxur may be explained mainly by the considerable loss of rat body weight on these diets, and not by the direct action of this pesticide on this organ. But the increase in the relative weight of kidney (and that of liver) seen in our experiments may be regarded as the result of the direct action of propoxur. Feron et al. (1973) found that when the rate of weight gain was reduced, the relative testicular weight decreased after the first 4 weeks of the experiment and increased after 13 weeks. In our investigations the significant rise in the relative weight of testicles after 93 days with propoxur concentration of 4500 ppm in the low-protein diet may be connected with the reduced gain body weight rate at this concentration of the pesticide, while the sudden fall of the relative testicular weight at 9000 ppm of propoxur with the same kind of diet was probably due to the direct toxic effect of the pesticide on this organ. Changes in the activity of the tested enzymes were similarly more pronounced when the animals received propoxur in the low-protein diet. These changes, although statistically significant, were small and their appearance at relatively high concentrations of propoxur (mostly at 4500 and 9000 ppm) indicates that they may be a threat to the organism, especially with a protein-deficient diet, only when high concentrations of propoxur are ingested during a long time period. The increased sensitivity of rats kept on diet insufficient in protein content to the toxic action of propoxur was more apparent when the preparation was given in a single dose through a gastric tube into the stomach than during long-term administration in diet. This sensitivity increased with longer administration of lowprotein diet. After 30 days the oral LD,, determined in rats on a low-protein diet was lower than in those kept on normoprotein diet 1.7 times in males and 1.3 times in females. After 93 days the LD,,, was lower, 4.3 times in males and 2.7 times in females, on a low-protein diet than in those on a normoprotein diet. In a paper published in 1969 Boyd and Stefec reported that the acute oral toxicity of 11 out of 12 pesticides studied in their laboratory increased two to eight times in rats kept for 28 days from weaning on a diet containing 3.5% of casein, as compared with rats kept on a diet with 26% of casein. The authors thought that this toxicity rise was not so important as to require investigations on humans for
PROPOXUR
IN
RATS
I61
establishing whether the use of these pesticides should be banned or limited in countries where the diet of the population is deficient in protein. It appears that this is also the case as regards the presently studied propoxur. This pesticide caused undesirable effects in rats when it was added to the diets chiefly in the highest concentrations and particularly after longer duration of the experiment. But it seems that propoxur, if properly handled in practice, should not penetrate into the organism in amounts capable of causing a harmful effect, especially if the diet of the population is, in general, deficient in protein. Finally, it is worth adding that we could not demonstrate a correlation between the level of biogenic amines and the activity of aromatic amino acids aminotransferases in the brain, although Gibb and Webb (1969) discussed the possibility that low activity of tyrosine aminotransferase in the brain suggests a fall in the level of biogenic amines there. In addition, it may be said that the decrease in the activity of aromatic amino acids aminotransferases in rat liver could be due to changes in the rhythm of food intake caused by propoxur or in the utilization of the diets given during the experiments. These factors could have caused alterations in the hepatic amino acid pool which regulates the activity of these enzymes (Rosen and Milholland, 1968; Fuller and Snoddy, 1968; Wurtman et al., 1968; Zigmond et al., 1969). The significantly lowered tyrosine transaminase activity was found by Century i 1972) in liver preparations from rats fed linseed or menhaden-oil-containing diets in comparison with animals fed low-polyunsaturated-fatty-acid-containing diets. The author stated that the significance of these results was not clear at that time and he did not suggest any probable mechanism for this relationship. Differences in the effects of propoxur on the transamination activity towards particular aromatic amino acids in the brain as well as in the liver support the view that different enzymes are responsible for transamination of these amino acids (Fonnum et al., 1964; Puiynska, 1972). REFERENCES Bernard, R. M., and Odell, L. D. (1950). Studies on beta-glucuronidase activity in pregnant albino rats. J. Luh. Clin. Med. 35, 940-944. Bertler. A. (1961). Effect of reserpine on the storage of catechol amines in brain and other tissues. Acra Physiol. Stand. St, 75-83. Bertler, A.. Carlsson, A.. and Rosengren. E. (1958). A method for the fluorimetric determination of adrenaline and noradrenaline in tissues. Aua Pllysiol. Scmzd. 44, 273-292. Boyd, E. M. (1961). Dietary protein and pesticide toxicity in male weanling rats. Bull. W. H. 0. 40, 801-805. Boyd, E. M.. Boulanger. M. A.. and De Castro, E. S. (1969). Phenacetin toxicity and dietary protein. Phurmacol. Res. Commun. 1, 15-19. Boyd, E. M., and Stefec, J. (1969). Dietary protein and pesticide toxicity. Cnud. Med. As.~oc~. J. 101, 33X-339. Cab Sci. 9, Part I]. l133- 1141. Singer, S., and Mason. M. (1965). Tyrosine-a-ketoglutarate transaminase. effect of the administration of sodium benzoate and related compounds on the hepatic enzyme level. Biochim. Biophys. Actrr 110, 370-379. Singer. S.. and Mason. M. (1967). The effects of the administration of sodium benzoate and diethylstilbestrol disulfate on the hepatic levels of several glucocorticoid-sensitive enzymes in adrenalectomired rats. &o&m. Birqhys. Acto. 146, 443-45 1. Singer. S.. and Mason. M. (1967). Studies of the in vitro and in vivo effects of conjugated steroids and carboxylic acids on hepatic tyrosine transaminase in the rat. Bioc.hirn. Biophvs. Acta 146, 451-466. Utley. J. D. (1963). The effects of anthranilic hydroxamic acid on rat behaviour and rat brain y-aminobutyric acid. norepinephrine and S-hydroxytryptamine concentrations. .I. Neurockm. 10, 423-431. Weatherholtz, W. M., Campbell, T. C.. and Webb, R. E. (1969). Effect of dietary protein levels on the toxicity and metabolism of heptachlor. J. N~itr. 98, 90-94. Wei]. C. S. (1953). Tables for convenient calculation of median-effective dose. LD,,, or ED,,,. and instructions in their use. Biomcrric,s 8, 249-163. Williams. C. H. (1969). p-glucuronidase activity in the serum and liver of rats administered pesticides and hepatotoxic agents. To.uiw[. Appl. Piwrmac~oi. 14, -783-29-7.
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IN
RATS
163
Wurtman. R. J., and Larin, F. (1968). A sensitive and specific isotopic assay for the estimation of tyrosine transaminase. Biochrn7. Pharmc7c~ol. 17, 817-818. Wurtman, R. J., Shoemaker. W. J.. and Larin. F. (1968). Mechanism of the daily rhythm in hepatic tyrosine transaminase activity: Role of dietary tryptophan. hoc. Nut. Acod. Sci. U.S.A. 59, 800-897. Zigmond, M. J.. Shoemaker. W. J., Larin. F.. and Wurtman, R. J. (1969). Hepatic tyrosine transaminase rhythm: Interaction of environmental lighting, food consumption and dietary protein content. .I. Ntrtr. 98. 71-75.
