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Short-term toxicity studies of rats fed triethyl phosphate in the diet
TOXICOLOGY
AND
Short-Term
APPLIED
Toxicity M.R.
12, 360-371
PHARMACOLOGY
(1968)
Studies of Rats in the Diet
Fed Triethyl
Phosphate
GUMBMANN, W. E. GAGNB, AND S.N.WILLIAMS
Western Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Albany, California 94710 Received November 8, I967
Triethyl phosphate (TEP) has potential for use as a whipping aid in commercially pasteurized egg whites; however, the Food and Drug Administration has not approved its use to date. Cunningham et al. (1967) have found that the whipping damage brought about by such processing may be effectively alleviated upon addition of TEP at a concentration of approximately 0.03 % or less. Unlike many other organophosphates, information on the metabolism of TEP is rather limited; understandably a result of the compound’s low toxicity and lack of detectable in vivo anticholinesterase activity. A sublethal intravenous dose of 1000 mg/kg of TEP in rats was found to produce deep anesthesia, but no cholinergic symptoms (Vandekar, 1957). For comparison, the lethal amount of malathion, which was classified as a weak cholinesterase inhibitor, was 600 mg/kg, with pronounced cholinergic symptoms evident at 300 mg/kg. Daviesetal. (1960) also found TEP to be nonneurotoxic when given to chickens intramuscularly at 50-100 mg/kg, as opposed to positive effects with diethyl phosphorofluoridate at 0.75 mg/kg. The LDss (iv.) to mice, reported by Cheymol et al. (1960) was 485 mg/kg. These workers found a cholinesterase inhibition of TEP in vitro amounting to 50% at a concentration of 50 pg/1.6 ml (about 1.7 x lo-4 M). However, diisopropyl phosphorofloridate, tetraethyl pyrophosphate, and paraoxon, for example, are one to ten thousand times more potent (Fleisher and Pope, 1954; DuBois and Mangun, 1947). This paper presents the biochemical and histologic findings of a study designed to determine the toxicity of TEP when fed to rats for 3 months. At the end of this time, reproduction studies were made. Included are the effects on two adaptive liver enzymes, tyrosine-c+ketoglutarate transaminase and alkaline phosphatase, and also brain and blood acetylcholinesterase, which have been shown to be responsive to acute poisoning by several cholinergic organophosphates (Murphy, 1966). METHODS
A preliminary 1Zday feeding trial with rats from our colony indicated that the maximum dietary concentration of TEP in Addis diet (Ambrose and Robbins, 1956) could not exceed 10 %. Food consumption decreased in excess of 60 % with 10 % TEP and about 45 % at 5 % TEP. With as little as 0.5 % TEP, food consumption was still noticeably lower. For the present investigation, Purina Laboratory Chow,1 used in our laboratory’s 1 Ralston-Purina Co., St. Louis, Missouri. approval or recommendation of the product of others that may be suitable.
Reference to a company by the U.S. Department 360
or product name does not imply of Agriculture to the exclusion
TOXICITY
OF TRIETHYL
PHOSPHATE
TO RATS
361
reproduction studies, was mixed with TEP to give five dietary concentrations : 0.1,0.5 1.O, 5.0, and 10 % by weight. Five Sprague-Dawley rats of each sex were used per level with an additional group of each sex fed only Purina Laboratory Chow to serve as controls. Initial average weight was 79 g for the males and 71 g for the females. The animals were maintained on the designated diets until autopsy. After 92 days, 3 males of each dietary group were mated one at a time for 1 week with females of the same group. The females were then caged individually until the young were weaned at the age of 21 days. The males were autopsied about day 120, and the females when the last litter was weaned at approximately 150 days. Food consumption of each group and individual body weights were determined weekly, including the weaning weights of the young. At 50 days, blood was obtained from the tail for determination of cholinesterase and packed cell volume, and again at 100 days for determination of cholinesterase, packed cell volume, hemoglobin concentration, and erythrocyte and leukocyte counts. At autopsy, etherized rats were killed by bleeding after blood samples had been obtained from the renal vein for blood urea nitrogen and plasma aIkaline phosphatase analyses. Complete necropsies were performed, and samples of abdominal and thoracic organs plus brain and pituitary were collected in 10% buffered, neutral formalin for histologic examination. One-half of the brain and the remaining liver were frozen in dry ice as soon as obtained and were held for later enzyme analyses. Red blood cell cholinesterase activity in whole blood was determined calorimetrically by the met hod of Fleisher et al. (1955) and adapted to tissue homogenates for the assay of brain and liver. Alkaline phosphatase was measured using phenyl phosphate as the substrate and the 4-amino antipyrine reagent with plasma and Ciocaiteu’s phenol reagent with liver (King and Wotton, 1956). Liver tyrosine transaminase was analyzed by the modification (Murphy, 1965) of the method of Kenney (1959). Blood urea nitrogen was determined by the procedure described by Annino (1964) and liver nitrogen by the Kjeldahl technique. Tissue sections were stained routinely with hematoxylin and eosin, and liver and kidney sections with Mallorys’ Reaction for Iron Stain (U.S. Armed Forces Institute of Pathology, 1960). TEP, practical grade, was obtained from Eastman Organic Chemicals2 and redistilled at atmospheric pressure before use, Statistical calculations included analysis of variance and Duncan’s multiple range test (Duncan, 1955) for comparison of group means and linear regression. RESULTS
Food Consumption and Growth In contrast to the preliminary feeding study in which food consumption was markedly reduced for 5 % TEP in Addis, Purina Rat Chow appeared to suffer no loss in palatability at this level (Table 1). At 10% TEP, however, food consumption was sharply reduced. Growth in general for both sexes was increasingly retarded with increasing concentration of TEP except for the males on 0.1% TEP, which outgrew all others by a small * Division of Eastman Kodak Company, Rochester, New York.
362
M. R. GUMBMANN,
W. E. GAGNI?,
AND
S. N. WILLIAMS
TABLE 1 FEED CONSUMPTION
Dietary level (%) ~__-Control 0.1
OF RATS FED TEP
FOR 92 DAYS
Mean feed consumption Wratldw) Males
Females _____
20.6 20.2 20.1 20.3 20.2
0.5
1.0 5.0
10.0
15.2 14.9 15.5 14.4 14.6 10.5"
11.8”
’ Meansare significantlydepressed below all other groups(P i 0.01). l-
- FEMALES - . . . . . . q CONTROL
400
-
40
60
80
DAYS FIG.
0
ON
.-.-
= 0.1%
TEP
m
= 0.5%
”
x----5:
= 1.0%
”
-
= 5.0%
”
-
= lo.oyo
‘1
*
= Rat
died
0
= Rat
autopsied
20
DIET
1. Growth of rats fed TEP.
40
60
80
100
TOXICITY
OF TRIETHYL
PHOSPHATE
363
TO RATS
margin (Fig. 1). At 10% TEP, the retardation was severe and compatible with decreased food consumption of that group, and several deaths occurred before 90 days. At 5 % TEP, body weight in the males became significantly depressed below the four lower groups by 29 days (P < 0.01). In the females at this dosage, body weight was depressed below the control and 0.1% TEP groups by 50 days (P < 0.05). Among the remaining groups, control through 1% TEP, no significant differences developed in either sex. Terminal
Body and Organ
Weights
The organ weight most noticeably affected was that of the liver (Table 2). Progressive increases especially in the males, occurred with increasing dietary concentrations of TEP through 5 %. At 10 % TEP in both sexes, where growth was considerably curtailed, liver weight was lower. In order to detect whether an alteration in basic composition might be associated with increased liver weight, liver nitrogen concentration on a fresh weight basis was determined. However, the latter was essentially constant for each group and without any significant differences. TABLE
2
TERMINAL MEAN BODY AND ORGAN WEIGHTS OF RATS FED TEP“
Dietary level (%> Control 0.1 0.5 1.0 5.0 lO.Ob
Control 0.1 0.5 1.0 5.0 10.Ob
Body weight
431k 432k 435k 430k 360’ 98
Liver
Kidneys
Males (120 Days) 14.4’ 2.96k 17.9’ 3.38” 21.4” 3.34k’ 21.0” 3.52’ 35.0” 3.20”
11.1
1.64
278k 265k 243k 257’ 176’
Females (150 Days) 12.1* 1.93k 15.0k 2.30k 15.1” 2.10k 15.6k 2.09k 23.1’ 2.00k
111
14.7
1.49
Spleen
Adrenals
0.79k 1.10’ o.84k 0.94k 0.90k 0.17
0.041k 0.051k 0.04Sk 0.057L 0.082’ 0.116’
0.66k 0.69k 0.63* O&IL 0.48’ 0.30
0.070k 0.075L 0.069k 0.072k 0.079L 0.163
a Weights in grams. Duncan’s multiple range test indicated by superscript letters k, I, m, and n; means with no superscript letter in common are significantly different at P < 0.05. b Values for the 10 % dietary level are based on two animals for the males and only one for the females and have not been included in any statistical analysis. ’ Individual adrenal weights for each animal : 0.082,O. 150.
Mean kidney weight of all groups, excluding 10% TEP was fairly uniform except for a significant increase in the 1% males. The relationship of this increased weight to TEP is not clear. 13
364
M. R. GUMBMANN,
W. E. GAGNB,
.4ND S. N. WILLIAMS
Spleen weight in the males appeared to be unaffected by TEP, excluding consideration of the underdeveloped rats at 10% TEP. The enlargement found in the 0.1% males does not seem to be related to TEP but rather to a respiratory infection this group of animals suffered around day 105. At this time, all five lost weight, and eventually two died. The remaining three recovered and returned to normal weight. In the females, spleen weight was lowered at 5 % TEP but was also a reflection of body weight as indicated by a highly significant correlation to body weight (PC 0.001, 22 degrees of freedom). Adrenal weight in the males became distinctly increased in response to TEP. Enlargement could be observed at 1% TEP, and at 5 % mean adrenal weight was significantly greater than that of all lower levels. Although not apparent in the mean adrenal weight of the females at 5 ‘A TEP, the same effect was noted in two animals at this level. Increased adrenal weight for the surviving animals of both sexes at 10% TEP was quite pronounced. Heart, lung, and brain weights in the females and heart weight in the males all closely reflected body weight as indicated by positive and highly significant correlations of these tissues to body weight. In the males, lung, brain, and testes weights were relatively independent of body weight and showed no significant response to TEP. Hematologic Findings
In the males fed 10 % TEP, the mean packed cell volume was depressed below that of all other diets at 50 days. However, by 100 days, slight hemoconcentration was indicated since packed cell volume, hemoglobin concentration, and erythrocyte count were all greatest in this group. At 5 y0 TEP the males had the lowest value of these three blood factors at 100 days, but significantly so only for erythrocyte count. In the females no significant hematologic differences were observed among the various groups. Leukocyte counts showed considerable variation without any significant differences between group means in either sex at 100 days. However, a relatively high count was observed for the 0.1% males which may have been the result of the aforementioned respiratory infection in this group. Blood Cholinesterase
Rats of both sexes fed intermediate levels of TEP showed small elevations in blood cholinesterase activity at 50 days (Table 3). At higher levels, 5 and 10% TEP for the males and 5% TEP for the females, enzyme activity was not significantly different from that of the controls. By 100 days, enzyme activity in the males had decreased in all groups, that of the 10% TEP group being significantly below all others. In the females at this time, activity had increased in the control and 0.1% TEP groups such that a progressive decrease in cholinesterase with increasing TEP dosage was evident. The single surviving female fed 10% TEP was an exception. However, this animal may be regarded as unusually resistant, since 4 others of this group had long since succumbed. Plasma Alkaline Phosphatase and Blood Urea Nitrogen
At autopsy, plasma alkaline phosphatase activity in both males and females was found to be depressed below control values at 1% TEP and also in the females at 5 %
365
TOXICITY OF TRIETHYL PHOSPHATE TO RATS
TABLE 3 BLOOD CHOLINESTERASE ACTIVITY
IN RATS
FED
TEP”
Cholinesteraseb Dietary level (%I
Females
Males 50 days
Control 0.1 0.5
79.0k 81.1k’ 87.81m 92.6m 75.Ok
1.0
5.0 10.0’
81 .2k’
100days
50 days
100days
69.7k 62.Sk 70.0k 67.9k 70.9k 37.8’
77.Y 89.8’ 93.1’ 89.4’ 82.4k’ 88.8
99.1k 95.6k’ 88.7’ 88.0’ 78.1” 80.8
aDuncan’smultiple range test indicated by superscript letters means with no superscript letter in common are significantly P
TEP (Table 4). The elevation in the males at 0.1% infection in this group 10-15 days earlier. Plasma females appeared to be as much influenced by lactation as by dietary TEP. Within each group,
k, I, and m; different at hydrolyzed animal and
TEP may be again the result of the alkaline phosphatase levels in the the occurrence of pregnancy and noticeably lower plasma alkaline
TABLE 4 TERMINAL
MEAN PLASMA UREA NITROGEN
ALKALINE PHOSPHATASE AND BLOOD LEVELS IN RATS FED TEP”
Males (120days) Dietary level (%) Control 0.1 0.5 1.0 5.0
Plasma alkaline phosphataseb (P < 0.01) 15.0k 20.6’ 17.8” l;:;;
Blood urea nitrogen” (PC 0.05) 21.7’ 18.5’ 19.0* 26.5’ 21.4k
Females(150days) Plasma alkaline phosphataseb (P cO.05) 22.1k 23.7k 21.2k’ 15.6’ 17.7’
Blood urea nitrogen”
(W 21.4 22.5 24.0 23.1 19.4
’ Duncan’s multiple range test indicated by superscript letters k, 1,and m; means with no superscript letter in common are significantly different at probability in parentheses. NS = not significant. b Expressed as King-Armstrong units per 100 ml of plasma. c Expressed as milligrams of urea N per 100 ml of blood.
366
M. R. GUMBMANN,
W. E. GAGNh,
AND
S. N. WILLIAMS
phosphatase activity was associated with those animals that had no young. Despite this influence, the significant depression in the 1 y0 TEP females seems related to diet, since in both this and the control group 4 out of 5 animals completed pregnancy. Blood urea nitrogen values were elevated in the males at 1% TEP, but not in the females, where there were no significant differences among the means of the various dietary groups. Liver
and Brain
Enzymes
at Autopsy
Liver alkaline phosphatase was significantly elevated at 5% TEP in both sexes (Table 5). The reason for the low mean in the males at 0.5 % TEP is not apparent. TABLE
5
LIVER AND BRAIN ENZYMES DETERMINED AT AUTOPSY IN RATS FED TEP”
Dietary level (%)
Liver alkaline phosphataseb
Liver tyrosine transaminaseC
Liver cholinesterascd
Brain cholinesteraaed
Males Control 0.1 0.5 1.0 5.0 Females Control 0.1 0.5 1.0 5.0
(P < 0.01) 0.519** 0.663’ 0.368’ 0.541k’ 0.952” (P < 0.05) 0.304* 0.203* 0.385” 0.312k 0.574’
(P -=z0.01) 12.38k 8.62’ 7.48rm 8.56’ 5.76” (P i 0.01) 12.77k 11.74k’ 9.72”” 8.91’” 6.80”
(P < 0.01) 33.0k 33.9k 23.9’ 26.5’ 32.6k O’S) 31.1 31.6 33.5 34.0 31.7
(P < 0.01) 699’ 618k’ 697* 61Sk’ 546’ W) 627 692 579 633 573
0 Duncan’s multiple range test indicated by superscript letters k, 1,and m; means with no superscript letter in common are significantly different at probability in parentheses. NS = not significant. b Expressed as King-Armstrong units per gram of fresh liver. c Expressed as micrograms of p-hydroxyphenylpyruvic acid formed per milligram of fresh liver per hour. d Expressed as micromoles of acetylcholine hydrolyzed per gram of fresh tissue per hour.
Liver tyrosine transaminase showed a tendency to become depressed with increasing TEP in both sexes. In the males, significant depression below the controls was evident in all groups and, in the females, in the two highest surviving groups, 1 and 5 % TEP. The correlation of liver tyrosine transaminase concentration to the reciprocal of liver weight was highly significant in both sexes (P -C0.001, 20 degrees of freedom), which is the relationship to be expected had the enzyme merely become increasingly diluted with increasing liver weight. Liver cholinesterase was significantly depressed in the males at 0.5 and 1% TEP, but not at 5 %. It remained unaffected in the females. Brain cholinesterase was lowest in both sexes at 5 % TEP, but only significantly so in the males.
TOXICITY
OF TRIETHYL
PHOSPHATE
367
TO RATS
Reproduction Summarized in Table 6 are reproduction and weaning data. Full-term pregnancies occurred in about the same frequency for each dietary group through 1% TEP; TABLE REPRODUCTIONOF
Dietary Number of level females mated (%) Control
5
Mean litter
Number of
litters
size at birth
cast dead
9.8
0 2
cast
0.5 1.0 5.0
z
10.0
1
0
5 4
RATSFEDTEP
Number of
4 4 3 4 0
0.1
6
PUPS
10.0
1
9.7 5.0 -
-
-
-
1
Mean litter sizeat weaning* 9.8 9.5 9.3 4.2 -
-
Mean body weight at weaning (g) 48.6 45.6 41.7 51.3
(49.6)* (46.7) (42.2) (46.2) -
-
aWeaningat 21days. * Mean of body weightscorrectedfor regression uponlitter size.
however, above this level growth and development were too retarded to permit successful mating. Litter size, which was markedly reduced at 1% TEP, greatly influenced body weight of the pups, as shown by a negative and highly significant correlation (P < 0.001, 120 degrees of freedom). Using this regression to adjust weaning weights to a constant litter size, a small depression in growth of the pups was evident for all groups receiving TEP. Histopathology
No significant microscopic lesions were found in rats of the 0.1 and 0.5 % TEP groups. Centrilobular hepatocytes of female rats in the 0.5 % TEP group were slightly larger, however, than comparable hepatocytes in the 0.1% and control groups. The initial lesion unequivocally produced by TEP occurred in the liver of female rats of the 1 ‘A TEP group. The lesion consisted of hypertrophic hepatocytes containing homogeneous darkly staining cytoplasm and enlarged nuclei with dense, thickened nuclear membranes. Nucleoli were also enlarged and more intensely basophilic than normal. Abnormal hepatocytes were confined to the centrilobular zone in four livers but involved all lobular regions of the fifth. Livers of male rats in the 1% TEP group were normal. Hepatocellular hypertrophy was slightly more extensive in the 5 % TEP group and occurred in both male and female rats. Enlarged hepatocytes were generally limited to centrilobular regions. The parenchyma of one liver however was entirely occupied by enlarged cells which were individually separated from each other (Fig. 2). In
368
M. R. GUMBMANN,
W. E. GAG&,
AND
S. N. WILLIAMS
addition, there was variable hepatocellular bile retention with minor bile stasis in canaliculi in all livers, and early bile ductule proliferation in three. Rare, isolated necrobiotic parenchymal cells were seen in most livers, and there was an occasional minute focus of necrosis situated close to foci of hemorrhage or congestion in two livers. Lipidosis occurred in one liver as multiple small fat vacuoles within swollen cytoplasm of cells adjacent to the centrilobular hypertrophic hepatocytes. Pigment comparable to bile retained in the liver was present in renal tubular epithelial cells of five rats. The pigment stained negatively for iron. Tissues were available only from three surviving rats, 2 males and 1 female, of the
FIG. 2. Hypertrophic hepatocytes with dissociation and bile ductule hyperplasia (arrow) in the liver of a rat fed 5 % TEP. Hematoxylin and eosin. x 30.
10% TEP group. Liver lesions were more severe in the males. There was generalized hepatocellular hypertrophy, extensive bile retention, and mild bile ductule hyperplasia. A few minute necrotic foci were present in one liver. Necrobiosis with nuclear pycnosis, karyorrhexis, and focal aggregation of chromatin on the nuclear membrane was more common and usually centrilobularly located in the second liver. There was also an accompanying loss of cellular alignment and minimal periportal fibrosis in a few instances. Hepatic lesions in the female rat were comparable with those of the 5% group. Renal tubular epithelial cells of all 3 rats in the 10 % TEP group contained pigment. In addition, there was extensive necrosis of the epithelium of most tubules occupying the cortex and the glomeruli in both kidneys from the female rat. There was no inflammatory reaction.
TOXICITY
OF TRIETHYL
PHOSPHATE
TO RATS
369
DISCUSSION Low toxicity of TEP ingested by rats was indicated in this investigation by biochemical changes which were, in many instances, not much greater than the minimum required for statistical detection and which as clinical indicators would be considered to be within normal ranges. More significant differences among the dietary groups were evident in the males, and some effects such as increased liver weight and decreased tyrosine transaminase reached to lower levels of TEP than in the females. Histologic changes, however, were generally more severe in the females. The reduction in food consumption at 10% TEP was such that the actual daily intake of TEP was not much greater than that of the 5% level. Consequently, the results of feeding 10% TEP cannot be attributed to TEP toxicity alone, but rather to the TEP plus the added stress of undernutrition. Thus growth suppression, which was severe at this level, could be clearly related to TEP only in the 5 y0 group. Uniform liver nitrogen concentration throughout all groups, excluding those at 10% TEP for which it was not determined, and microscopic examination indicated that cellular hypertrophy and the resulting increases in liver weight were not the result of such changes as increased water content or fatty infiltration, but were apparently compensatory in nature. Adrenal enlargement was also an apparent response to the stress of TEP. The hematologic findings were unremarkable, except for a slight but significantly lowered erythrocyte count in the 5 % males. Adaptation to the low anticholinesterase activity of TEP, detected in vitro by Cheymol et al. (1960), may have occurred from the continual feeding of TEP. A temporary, small elevation in blood cholinesterase activity was detected at 50 days in both sexes at the 0.5 and 1% TEP levels and also in the females at the 0.1% level. At autopsy, decreased liver cholinesterase activity was present for the same two groups of males. Blood cholinesterase in the control and 0.1% TEP females, unlike that of the males, increased with time. However, at 0.5 y0 TEP or more, this increase was not observed. Brain cholinesterase in both sexes tended to be decreased at 5 % TEP, which is similar to the effect of acute doses of known cholinesterase inhibitors (Murphy, 1966). Liver alterations capable of increasing plasma alkaline phosphatase were absent, even at 5 % TEP. Instead, plasma alkaline phosphatase became lower at higher TEP levels. Although pigment was observed in renal tubular epithelial cells of the 5 % TEP group, there appeared to be no general impairment of kidney function as shown by the absence of elevated blood urea nitrogen except in the 1 ‘A TEP males. The significance of the latter in regard to TEP is not certain. Murphy (1966) has shown that acute administration of toxic agents, including several cholinergic organophosphates, is capable of increasing rat liver alkaline phosphatase and tyrosine transaminase. This effect is probably mediated through the pituitary-adrenal system by a general stress mechanism. In the case of subacute feeding of TEP, only liver alkaline phosphatase became elevated and only at 5 % TEP. Conversely, the concentration of liver tyrosine transaminase activity was significantly lowered for all groups of males receiving TEP and in the females at 1% and 5 % TEP. Thus, in contrast to nitrogen constituents in general, production of this enzyme failed
370
M. R. GUMBMANN,
W.
E. GAGN6,
AND
S. N.
WILLIAMS
to keep pace with increasing liver weight, and total activity tended to remain more or less constant. Reproduction was adversely affected at 1% TEP and completely prevented at 5 % TEP. Growth of the young was slightly retarded in all groups receiving TEP. The primary histologic response to TEP was compensatory hepatocellular hypertrophy followed by minor bile ductule hyperplasia and retention of bile. The lesions were generally mild and only relatively severe in the 10% group. Since consumption of TEP in this group compared with the 5% TEP group, the degenerative hepatic lesions, necrobiosis, pycnosis, karyorrhexis, and focal aggregation of chromatin were probably related to malnutrition rather than TEP alone. SUMMARY Triethylphosphate (TEP) was fed to rats at dietary levels of 0.1, 0.5, 1.O, 5.0, and 10 % for 3 months plus additional time for reproduction studies. Inhibition of growth related to TEP occurred at the 5 ‘A level, and reproduction was adversely affected at 1 ‘A. Severe anorexia at 10 % TEP made it necessary to exclude this group from consideration of TEP toxicity. Liver weight was markedly increased, being detectable in the males at 0.1% TEP or more and in the females at 5 % TEP. Adrenal enlargement also occurred at the highest levels, especially in the males. Clinical studies included measurements of blood, brain, and liver acetylcholinesterase, plasma and liver alkaline phosphatase, liver tyrosine transaminase, blood urea nitrogen, liver nitrogen, and hematology. Alterations of these factors were for the most part minimal and would be considered to be within normal ranges. Small, transient elevations of blood cholinesterase were detected at 50 days, followed at 100 days by slight depression in the females receiving 0.5 % TEP or more. Brain cholinesterase was also slightly depressed at 5 % TEP. Liver alkaline phosphatase was elevated at 5% TEP, while liver tyrosine transaminase became significantly depressed in the males at 0.1% or more and in the females at 5 %. Hepatocellular enlargement developed initially in the 1% female rats and appeared in both male and female rats of the 5 % group. Minor bile ductule hyperplasia and retention of bile also occurred in the latter group. At levels that would be encountered if used as proposed as a food additive, the daily dosage of TEP would be far below the no-effect level in the rat as related to the present study. REFERENCES A. M., and ROBBINS, D. J. (1956). Studies on comparative absorption and digestibility of acetoglycerides. J. Nutr. 58, 113-124. ANNINO, J. S. (1964). Clinical Chemistry; Principles and Procedures, 3rd ed., pp. 154-160. Little, Brown, Boston, Massachusetts. CHEYMOL, J., CHABRIER, P., MURAD,J., and SELIM, M. (1960).Contribution a l’etudechimique et pharmacologiquedesderivesde l’acide orthophosphorique(III). Constitution chimique et activite anticholinesterasique.Therapie 15, 237-241. CUNNINGHAM, F. E., KLINE, L., andLINEWEAVER, H. (1967).Eggwhite compositioncontaining triethyl phosphatehaving enhancedwhipping properties.U.S. Patent 3,328,175,June 27. DAWES, D. R., HOLLAND, P., andRUMENS, M. J. (1960).The relationshipbetweenthe chemical structure and neurotoxicity of alkyl organophosphoruscompounds.Brit. J. Pharmacol. 15, 271-278. DUBOIS,K. P., and MANGUN, G. H. (1947). Effect of hexaethyl tetraphosphateon choline esterasein vitro and in vivo. Proc. Sot. Exptl. Biol. Med. 64, 137-139. DUNCAN, D. B. (1955).Multiple rangeand multiple F. tests.Biometrics 11, l-42. FLEISHER, J. H., and POPE, E. J. (1954).Calorimetric method for determinationof red blood cell cholinesterase activity in whole blood. Arch. Znd. Hyg. Occupational Med. 9, 323-334. AMFIROSE,
TOXICITY OF TRIETHYL PHOSPHATE TO RATS
371
FLEISHER,J. H., POPE,E. J., and SPEAR, S. F. (1955). Determination of red blood cell cholinesterase activity in whole blood. Arch. Ind. Health 11, 332-337. KENNEY,F. T. (1959).Propertiesof partially purified tyrosine-a-ketoglutaratetransaminase from rat liver. J. Biol. Chem. 234,2707-2712. KING, E. J., and WOTTON, I. D. P. (1956).Micro-analysis in Medical Biochemistry, 3rd ed., pp. 81-86. Grune & Stratton, New York. MURPHY, S.D. (1965).Mechanismof the effect of acrolein on rat liver enzymes.Toxicol. Appl. Pharmacol.
7,833-843.
MURPHY,S. D.(1966). Responseof adaptive rat liver enzymesto acutepoisoningby organophosphateinsecticides.Toxicol. Appl. Pharmacol. 8,266-276. U.S. Armed ForcesInstitute of Pathology (1960).Manual of Histologic and Special Staining Technics, 2nd ed., pp. 149-150.McGraw-Hill, New York. VANDEKAR,M. (1957).Anesthetic effect producedby organopbosphorus compounds.Nature 179, 154-l 55.
AND
Short-Term
APPLIED
Toxicity M.R.
12, 360-371
PHARMACOLOGY
(1968)
Studies of Rats in the Diet
Fed Triethyl
Phosphate
GUMBMANN, W. E. GAGNB, AND S.N.WILLIAMS
Western Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Albany, California 94710 Received November 8, I967
Triethyl phosphate (TEP) has potential for use as a whipping aid in commercially pasteurized egg whites; however, the Food and Drug Administration has not approved its use to date. Cunningham et al. (1967) have found that the whipping damage brought about by such processing may be effectively alleviated upon addition of TEP at a concentration of approximately 0.03 % or less. Unlike many other organophosphates, information on the metabolism of TEP is rather limited; understandably a result of the compound’s low toxicity and lack of detectable in vivo anticholinesterase activity. A sublethal intravenous dose of 1000 mg/kg of TEP in rats was found to produce deep anesthesia, but no cholinergic symptoms (Vandekar, 1957). For comparison, the lethal amount of malathion, which was classified as a weak cholinesterase inhibitor, was 600 mg/kg, with pronounced cholinergic symptoms evident at 300 mg/kg. Daviesetal. (1960) also found TEP to be nonneurotoxic when given to chickens intramuscularly at 50-100 mg/kg, as opposed to positive effects with diethyl phosphorofluoridate at 0.75 mg/kg. The LDss (iv.) to mice, reported by Cheymol et al. (1960) was 485 mg/kg. These workers found a cholinesterase inhibition of TEP in vitro amounting to 50% at a concentration of 50 pg/1.6 ml (about 1.7 x lo-4 M). However, diisopropyl phosphorofloridate, tetraethyl pyrophosphate, and paraoxon, for example, are one to ten thousand times more potent (Fleisher and Pope, 1954; DuBois and Mangun, 1947). This paper presents the biochemical and histologic findings of a study designed to determine the toxicity of TEP when fed to rats for 3 months. At the end of this time, reproduction studies were made. Included are the effects on two adaptive liver enzymes, tyrosine-c+ketoglutarate transaminase and alkaline phosphatase, and also brain and blood acetylcholinesterase, which have been shown to be responsive to acute poisoning by several cholinergic organophosphates (Murphy, 1966). METHODS
A preliminary 1Zday feeding trial with rats from our colony indicated that the maximum dietary concentration of TEP in Addis diet (Ambrose and Robbins, 1956) could not exceed 10 %. Food consumption decreased in excess of 60 % with 10 % TEP and about 45 % at 5 % TEP. With as little as 0.5 % TEP, food consumption was still noticeably lower. For the present investigation, Purina Laboratory Chow,1 used in our laboratory’s 1 Ralston-Purina Co., St. Louis, Missouri. approval or recommendation of the product of others that may be suitable.
Reference to a company by the U.S. Department 360
or product name does not imply of Agriculture to the exclusion
TOXICITY
OF TRIETHYL
PHOSPHATE
TO RATS
361
reproduction studies, was mixed with TEP to give five dietary concentrations : 0.1,0.5 1.O, 5.0, and 10 % by weight. Five Sprague-Dawley rats of each sex were used per level with an additional group of each sex fed only Purina Laboratory Chow to serve as controls. Initial average weight was 79 g for the males and 71 g for the females. The animals were maintained on the designated diets until autopsy. After 92 days, 3 males of each dietary group were mated one at a time for 1 week with females of the same group. The females were then caged individually until the young were weaned at the age of 21 days. The males were autopsied about day 120, and the females when the last litter was weaned at approximately 150 days. Food consumption of each group and individual body weights were determined weekly, including the weaning weights of the young. At 50 days, blood was obtained from the tail for determination of cholinesterase and packed cell volume, and again at 100 days for determination of cholinesterase, packed cell volume, hemoglobin concentration, and erythrocyte and leukocyte counts. At autopsy, etherized rats were killed by bleeding after blood samples had been obtained from the renal vein for blood urea nitrogen and plasma aIkaline phosphatase analyses. Complete necropsies were performed, and samples of abdominal and thoracic organs plus brain and pituitary were collected in 10% buffered, neutral formalin for histologic examination. One-half of the brain and the remaining liver were frozen in dry ice as soon as obtained and were held for later enzyme analyses. Red blood cell cholinesterase activity in whole blood was determined calorimetrically by the met hod of Fleisher et al. (1955) and adapted to tissue homogenates for the assay of brain and liver. Alkaline phosphatase was measured using phenyl phosphate as the substrate and the 4-amino antipyrine reagent with plasma and Ciocaiteu’s phenol reagent with liver (King and Wotton, 1956). Liver tyrosine transaminase was analyzed by the modification (Murphy, 1965) of the method of Kenney (1959). Blood urea nitrogen was determined by the procedure described by Annino (1964) and liver nitrogen by the Kjeldahl technique. Tissue sections were stained routinely with hematoxylin and eosin, and liver and kidney sections with Mallorys’ Reaction for Iron Stain (U.S. Armed Forces Institute of Pathology, 1960). TEP, practical grade, was obtained from Eastman Organic Chemicals2 and redistilled at atmospheric pressure before use, Statistical calculations included analysis of variance and Duncan’s multiple range test (Duncan, 1955) for comparison of group means and linear regression. RESULTS
Food Consumption and Growth In contrast to the preliminary feeding study in which food consumption was markedly reduced for 5 % TEP in Addis, Purina Rat Chow appeared to suffer no loss in palatability at this level (Table 1). At 10% TEP, however, food consumption was sharply reduced. Growth in general for both sexes was increasingly retarded with increasing concentration of TEP except for the males on 0.1% TEP, which outgrew all others by a small * Division of Eastman Kodak Company, Rochester, New York.
362
M. R. GUMBMANN,
W. E. GAGNI?,
AND
S. N. WILLIAMS
TABLE 1 FEED CONSUMPTION
Dietary level (%) ~__-Control 0.1
OF RATS FED TEP
FOR 92 DAYS
Mean feed consumption Wratldw) Males
Females _____
20.6 20.2 20.1 20.3 20.2
0.5
1.0 5.0
10.0
15.2 14.9 15.5 14.4 14.6 10.5"
11.8”
’ Meansare significantlydepressed below all other groups(P i 0.01). l-
- FEMALES - . . . . . . q CONTROL
400
-
40
60
80
DAYS FIG.
0
ON
.-.-
= 0.1%
TEP
m
= 0.5%
”
x----5:
= 1.0%
”
-
= 5.0%
”
-
= lo.oyo
‘1
*
= Rat
died
0
= Rat
autopsied
20
DIET
1. Growth of rats fed TEP.
40
60
80
100
TOXICITY
OF TRIETHYL
PHOSPHATE
363
TO RATS
margin (Fig. 1). At 10% TEP, the retardation was severe and compatible with decreased food consumption of that group, and several deaths occurred before 90 days. At 5 % TEP, body weight in the males became significantly depressed below the four lower groups by 29 days (P < 0.01). In the females at this dosage, body weight was depressed below the control and 0.1% TEP groups by 50 days (P < 0.05). Among the remaining groups, control through 1% TEP, no significant differences developed in either sex. Terminal
Body and Organ
Weights
The organ weight most noticeably affected was that of the liver (Table 2). Progressive increases especially in the males, occurred with increasing dietary concentrations of TEP through 5 %. At 10 % TEP in both sexes, where growth was considerably curtailed, liver weight was lower. In order to detect whether an alteration in basic composition might be associated with increased liver weight, liver nitrogen concentration on a fresh weight basis was determined. However, the latter was essentially constant for each group and without any significant differences. TABLE
2
TERMINAL MEAN BODY AND ORGAN WEIGHTS OF RATS FED TEP“
Dietary level (%> Control 0.1 0.5 1.0 5.0 lO.Ob
Control 0.1 0.5 1.0 5.0 10.Ob
Body weight
431k 432k 435k 430k 360’ 98
Liver
Kidneys
Males (120 Days) 14.4’ 2.96k 17.9’ 3.38” 21.4” 3.34k’ 21.0” 3.52’ 35.0” 3.20”
11.1
1.64
278k 265k 243k 257’ 176’
Females (150 Days) 12.1* 1.93k 15.0k 2.30k 15.1” 2.10k 15.6k 2.09k 23.1’ 2.00k
111
14.7
1.49
Spleen
Adrenals
0.79k 1.10’ o.84k 0.94k 0.90k 0.17
0.041k 0.051k 0.04Sk 0.057L 0.082’ 0.116’
0.66k 0.69k 0.63* O&IL 0.48’ 0.30
0.070k 0.075L 0.069k 0.072k 0.079L 0.163
a Weights in grams. Duncan’s multiple range test indicated by superscript letters k, I, m, and n; means with no superscript letter in common are significantly different at P < 0.05. b Values for the 10 % dietary level are based on two animals for the males and only one for the females and have not been included in any statistical analysis. ’ Individual adrenal weights for each animal : 0.082,O. 150.
Mean kidney weight of all groups, excluding 10% TEP was fairly uniform except for a significant increase in the 1% males. The relationship of this increased weight to TEP is not clear. 13
364
M. R. GUMBMANN,
W. E. GAGNB,
.4ND S. N. WILLIAMS
Spleen weight in the males appeared to be unaffected by TEP, excluding consideration of the underdeveloped rats at 10% TEP. The enlargement found in the 0.1% males does not seem to be related to TEP but rather to a respiratory infection this group of animals suffered around day 105. At this time, all five lost weight, and eventually two died. The remaining three recovered and returned to normal weight. In the females, spleen weight was lowered at 5 % TEP but was also a reflection of body weight as indicated by a highly significant correlation to body weight (PC 0.001, 22 degrees of freedom). Adrenal weight in the males became distinctly increased in response to TEP. Enlargement could be observed at 1% TEP, and at 5 % mean adrenal weight was significantly greater than that of all lower levels. Although not apparent in the mean adrenal weight of the females at 5 ‘A TEP, the same effect was noted in two animals at this level. Increased adrenal weight for the surviving animals of both sexes at 10% TEP was quite pronounced. Heart, lung, and brain weights in the females and heart weight in the males all closely reflected body weight as indicated by positive and highly significant correlations of these tissues to body weight. In the males, lung, brain, and testes weights were relatively independent of body weight and showed no significant response to TEP. Hematologic Findings
In the males fed 10 % TEP, the mean packed cell volume was depressed below that of all other diets at 50 days. However, by 100 days, slight hemoconcentration was indicated since packed cell volume, hemoglobin concentration, and erythrocyte count were all greatest in this group. At 5 y0 TEP the males had the lowest value of these three blood factors at 100 days, but significantly so only for erythrocyte count. In the females no significant hematologic differences were observed among the various groups. Leukocyte counts showed considerable variation without any significant differences between group means in either sex at 100 days. However, a relatively high count was observed for the 0.1% males which may have been the result of the aforementioned respiratory infection in this group. Blood Cholinesterase
Rats of both sexes fed intermediate levels of TEP showed small elevations in blood cholinesterase activity at 50 days (Table 3). At higher levels, 5 and 10% TEP for the males and 5% TEP for the females, enzyme activity was not significantly different from that of the controls. By 100 days, enzyme activity in the males had decreased in all groups, that of the 10% TEP group being significantly below all others. In the females at this time, activity had increased in the control and 0.1% TEP groups such that a progressive decrease in cholinesterase with increasing TEP dosage was evident. The single surviving female fed 10% TEP was an exception. However, this animal may be regarded as unusually resistant, since 4 others of this group had long since succumbed. Plasma Alkaline Phosphatase and Blood Urea Nitrogen
At autopsy, plasma alkaline phosphatase activity in both males and females was found to be depressed below control values at 1% TEP and also in the females at 5 %
365
TOXICITY OF TRIETHYL PHOSPHATE TO RATS
TABLE 3 BLOOD CHOLINESTERASE ACTIVITY
IN RATS
FED
TEP”
Cholinesteraseb Dietary level (%I
Females
Males 50 days
Control 0.1 0.5
79.0k 81.1k’ 87.81m 92.6m 75.Ok
1.0
5.0 10.0’
81 .2k’
100days
50 days
100days
69.7k 62.Sk 70.0k 67.9k 70.9k 37.8’
77.Y 89.8’ 93.1’ 89.4’ 82.4k’ 88.8
99.1k 95.6k’ 88.7’ 88.0’ 78.1” 80.8
aDuncan’smultiple range test indicated by superscript letters means with no superscript letter in common are significantly P
TEP (Table 4). The elevation in the males at 0.1% infection in this group 10-15 days earlier. Plasma females appeared to be as much influenced by lactation as by dietary TEP. Within each group,
k, I, and m; different at hydrolyzed animal and
TEP may be again the result of the alkaline phosphatase levels in the the occurrence of pregnancy and noticeably lower plasma alkaline
TABLE 4 TERMINAL
MEAN PLASMA UREA NITROGEN
ALKALINE PHOSPHATASE AND BLOOD LEVELS IN RATS FED TEP”
Males (120days) Dietary level (%) Control 0.1 0.5 1.0 5.0
Plasma alkaline phosphataseb (P < 0.01) 15.0k 20.6’ 17.8” l;:;;
Blood urea nitrogen” (PC 0.05) 21.7’ 18.5’ 19.0* 26.5’ 21.4k
Females(150days) Plasma alkaline phosphataseb (P cO.05) 22.1k 23.7k 21.2k’ 15.6’ 17.7’
Blood urea nitrogen”
(W 21.4 22.5 24.0 23.1 19.4
’ Duncan’s multiple range test indicated by superscript letters k, 1,and m; means with no superscript letter in common are significantly different at probability in parentheses. NS = not significant. b Expressed as King-Armstrong units per 100 ml of plasma. c Expressed as milligrams of urea N per 100 ml of blood.
366
M. R. GUMBMANN,
W. E. GAGNh,
AND
S. N. WILLIAMS
phosphatase activity was associated with those animals that had no young. Despite this influence, the significant depression in the 1 y0 TEP females seems related to diet, since in both this and the control group 4 out of 5 animals completed pregnancy. Blood urea nitrogen values were elevated in the males at 1% TEP, but not in the females, where there were no significant differences among the means of the various dietary groups. Liver
and Brain
Enzymes
at Autopsy
Liver alkaline phosphatase was significantly elevated at 5% TEP in both sexes (Table 5). The reason for the low mean in the males at 0.5 % TEP is not apparent. TABLE
5
LIVER AND BRAIN ENZYMES DETERMINED AT AUTOPSY IN RATS FED TEP”
Dietary level (%)
Liver alkaline phosphataseb
Liver tyrosine transaminaseC
Liver cholinesterascd
Brain cholinesteraaed
Males Control 0.1 0.5 1.0 5.0 Females Control 0.1 0.5 1.0 5.0
(P < 0.01) 0.519** 0.663’ 0.368’ 0.541k’ 0.952” (P < 0.05) 0.304* 0.203* 0.385” 0.312k 0.574’
(P -=z0.01) 12.38k 8.62’ 7.48rm 8.56’ 5.76” (P i 0.01) 12.77k 11.74k’ 9.72”” 8.91’” 6.80”
(P < 0.01) 33.0k 33.9k 23.9’ 26.5’ 32.6k O’S) 31.1 31.6 33.5 34.0 31.7
(P < 0.01) 699’ 618k’ 697* 61Sk’ 546’ W) 627 692 579 633 573
0 Duncan’s multiple range test indicated by superscript letters k, 1,and m; means with no superscript letter in common are significantly different at probability in parentheses. NS = not significant. b Expressed as King-Armstrong units per gram of fresh liver. c Expressed as micrograms of p-hydroxyphenylpyruvic acid formed per milligram of fresh liver per hour. d Expressed as micromoles of acetylcholine hydrolyzed per gram of fresh tissue per hour.
Liver tyrosine transaminase showed a tendency to become depressed with increasing TEP in both sexes. In the males, significant depression below the controls was evident in all groups and, in the females, in the two highest surviving groups, 1 and 5 % TEP. The correlation of liver tyrosine transaminase concentration to the reciprocal of liver weight was highly significant in both sexes (P -C0.001, 20 degrees of freedom), which is the relationship to be expected had the enzyme merely become increasingly diluted with increasing liver weight. Liver cholinesterase was significantly depressed in the males at 0.5 and 1% TEP, but not at 5 %. It remained unaffected in the females. Brain cholinesterase was lowest in both sexes at 5 % TEP, but only significantly so in the males.
TOXICITY
OF TRIETHYL
PHOSPHATE
367
TO RATS
Reproduction Summarized in Table 6 are reproduction and weaning data. Full-term pregnancies occurred in about the same frequency for each dietary group through 1% TEP; TABLE REPRODUCTIONOF
Dietary Number of level females mated (%) Control
5
Mean litter
Number of
litters
size at birth
cast dead
9.8
0 2
cast
0.5 1.0 5.0
z
10.0
1
0
5 4
RATSFEDTEP
Number of
4 4 3 4 0
0.1
6
PUPS
10.0
1
9.7 5.0 -
-
-
-
1
Mean litter sizeat weaning* 9.8 9.5 9.3 4.2 -
-
Mean body weight at weaning (g) 48.6 45.6 41.7 51.3
(49.6)* (46.7) (42.2) (46.2) -
-
aWeaningat 21days. * Mean of body weightscorrectedfor regression uponlitter size.
however, above this level growth and development were too retarded to permit successful mating. Litter size, which was markedly reduced at 1% TEP, greatly influenced body weight of the pups, as shown by a negative and highly significant correlation (P < 0.001, 120 degrees of freedom). Using this regression to adjust weaning weights to a constant litter size, a small depression in growth of the pups was evident for all groups receiving TEP. Histopathology
No significant microscopic lesions were found in rats of the 0.1 and 0.5 % TEP groups. Centrilobular hepatocytes of female rats in the 0.5 % TEP group were slightly larger, however, than comparable hepatocytes in the 0.1% and control groups. The initial lesion unequivocally produced by TEP occurred in the liver of female rats of the 1 ‘A TEP group. The lesion consisted of hypertrophic hepatocytes containing homogeneous darkly staining cytoplasm and enlarged nuclei with dense, thickened nuclear membranes. Nucleoli were also enlarged and more intensely basophilic than normal. Abnormal hepatocytes were confined to the centrilobular zone in four livers but involved all lobular regions of the fifth. Livers of male rats in the 1% TEP group were normal. Hepatocellular hypertrophy was slightly more extensive in the 5 % TEP group and occurred in both male and female rats. Enlarged hepatocytes were generally limited to centrilobular regions. The parenchyma of one liver however was entirely occupied by enlarged cells which were individually separated from each other (Fig. 2). In
368
M. R. GUMBMANN,
W. E. GAG&,
AND
S. N. WILLIAMS
addition, there was variable hepatocellular bile retention with minor bile stasis in canaliculi in all livers, and early bile ductule proliferation in three. Rare, isolated necrobiotic parenchymal cells were seen in most livers, and there was an occasional minute focus of necrosis situated close to foci of hemorrhage or congestion in two livers. Lipidosis occurred in one liver as multiple small fat vacuoles within swollen cytoplasm of cells adjacent to the centrilobular hypertrophic hepatocytes. Pigment comparable to bile retained in the liver was present in renal tubular epithelial cells of five rats. The pigment stained negatively for iron. Tissues were available only from three surviving rats, 2 males and 1 female, of the
FIG. 2. Hypertrophic hepatocytes with dissociation and bile ductule hyperplasia (arrow) in the liver of a rat fed 5 % TEP. Hematoxylin and eosin. x 30.
10% TEP group. Liver lesions were more severe in the males. There was generalized hepatocellular hypertrophy, extensive bile retention, and mild bile ductule hyperplasia. A few minute necrotic foci were present in one liver. Necrobiosis with nuclear pycnosis, karyorrhexis, and focal aggregation of chromatin on the nuclear membrane was more common and usually centrilobularly located in the second liver. There was also an accompanying loss of cellular alignment and minimal periportal fibrosis in a few instances. Hepatic lesions in the female rat were comparable with those of the 5% group. Renal tubular epithelial cells of all 3 rats in the 10 % TEP group contained pigment. In addition, there was extensive necrosis of the epithelium of most tubules occupying the cortex and the glomeruli in both kidneys from the female rat. There was no inflammatory reaction.
TOXICITY
OF TRIETHYL
PHOSPHATE
TO RATS
369
DISCUSSION Low toxicity of TEP ingested by rats was indicated in this investigation by biochemical changes which were, in many instances, not much greater than the minimum required for statistical detection and which as clinical indicators would be considered to be within normal ranges. More significant differences among the dietary groups were evident in the males, and some effects such as increased liver weight and decreased tyrosine transaminase reached to lower levels of TEP than in the females. Histologic changes, however, were generally more severe in the females. The reduction in food consumption at 10% TEP was such that the actual daily intake of TEP was not much greater than that of the 5% level. Consequently, the results of feeding 10% TEP cannot be attributed to TEP toxicity alone, but rather to the TEP plus the added stress of undernutrition. Thus growth suppression, which was severe at this level, could be clearly related to TEP only in the 5 y0 group. Uniform liver nitrogen concentration throughout all groups, excluding those at 10% TEP for which it was not determined, and microscopic examination indicated that cellular hypertrophy and the resulting increases in liver weight were not the result of such changes as increased water content or fatty infiltration, but were apparently compensatory in nature. Adrenal enlargement was also an apparent response to the stress of TEP. The hematologic findings were unremarkable, except for a slight but significantly lowered erythrocyte count in the 5 % males. Adaptation to the low anticholinesterase activity of TEP, detected in vitro by Cheymol et al. (1960), may have occurred from the continual feeding of TEP. A temporary, small elevation in blood cholinesterase activity was detected at 50 days in both sexes at the 0.5 and 1% TEP levels and also in the females at the 0.1% level. At autopsy, decreased liver cholinesterase activity was present for the same two groups of males. Blood cholinesterase in the control and 0.1% TEP females, unlike that of the males, increased with time. However, at 0.5 y0 TEP or more, this increase was not observed. Brain cholinesterase in both sexes tended to be decreased at 5 % TEP, which is similar to the effect of acute doses of known cholinesterase inhibitors (Murphy, 1966). Liver alterations capable of increasing plasma alkaline phosphatase were absent, even at 5 % TEP. Instead, plasma alkaline phosphatase became lower at higher TEP levels. Although pigment was observed in renal tubular epithelial cells of the 5 % TEP group, there appeared to be no general impairment of kidney function as shown by the absence of elevated blood urea nitrogen except in the 1 ‘A TEP males. The significance of the latter in regard to TEP is not certain. Murphy (1966) has shown that acute administration of toxic agents, including several cholinergic organophosphates, is capable of increasing rat liver alkaline phosphatase and tyrosine transaminase. This effect is probably mediated through the pituitary-adrenal system by a general stress mechanism. In the case of subacute feeding of TEP, only liver alkaline phosphatase became elevated and only at 5 % TEP. Conversely, the concentration of liver tyrosine transaminase activity was significantly lowered for all groups of males receiving TEP and in the females at 1% and 5 % TEP. Thus, in contrast to nitrogen constituents in general, production of this enzyme failed
370
M. R. GUMBMANN,
W.
E. GAGN6,
AND
S. N.
WILLIAMS
to keep pace with increasing liver weight, and total activity tended to remain more or less constant. Reproduction was adversely affected at 1% TEP and completely prevented at 5 % TEP. Growth of the young was slightly retarded in all groups receiving TEP. The primary histologic response to TEP was compensatory hepatocellular hypertrophy followed by minor bile ductule hyperplasia and retention of bile. The lesions were generally mild and only relatively severe in the 10% group. Since consumption of TEP in this group compared with the 5% TEP group, the degenerative hepatic lesions, necrobiosis, pycnosis, karyorrhexis, and focal aggregation of chromatin were probably related to malnutrition rather than TEP alone. SUMMARY Triethylphosphate (TEP) was fed to rats at dietary levels of 0.1, 0.5, 1.O, 5.0, and 10 % for 3 months plus additional time for reproduction studies. Inhibition of growth related to TEP occurred at the 5 ‘A level, and reproduction was adversely affected at 1 ‘A. Severe anorexia at 10 % TEP made it necessary to exclude this group from consideration of TEP toxicity. Liver weight was markedly increased, being detectable in the males at 0.1% TEP or more and in the females at 5 % TEP. Adrenal enlargement also occurred at the highest levels, especially in the males. Clinical studies included measurements of blood, brain, and liver acetylcholinesterase, plasma and liver alkaline phosphatase, liver tyrosine transaminase, blood urea nitrogen, liver nitrogen, and hematology. Alterations of these factors were for the most part minimal and would be considered to be within normal ranges. Small, transient elevations of blood cholinesterase were detected at 50 days, followed at 100 days by slight depression in the females receiving 0.5 % TEP or more. Brain cholinesterase was also slightly depressed at 5 % TEP. Liver alkaline phosphatase was elevated at 5% TEP, while liver tyrosine transaminase became significantly depressed in the males at 0.1% or more and in the females at 5 %. Hepatocellular enlargement developed initially in the 1% female rats and appeared in both male and female rats of the 5 % group. Minor bile ductule hyperplasia and retention of bile also occurred in the latter group. At levels that would be encountered if used as proposed as a food additive, the daily dosage of TEP would be far below the no-effect level in the rat as related to the present study. REFERENCES A. M., and ROBBINS, D. J. (1956). Studies on comparative absorption and digestibility of acetoglycerides. J. Nutr. 58, 113-124. ANNINO, J. S. (1964). Clinical Chemistry; Principles and Procedures, 3rd ed., pp. 154-160. Little, Brown, Boston, Massachusetts. CHEYMOL, J., CHABRIER, P., MURAD,J., and SELIM, M. (1960).Contribution a l’etudechimique et pharmacologiquedesderivesde l’acide orthophosphorique(III). Constitution chimique et activite anticholinesterasique.Therapie 15, 237-241. CUNNINGHAM, F. E., KLINE, L., andLINEWEAVER, H. (1967).Eggwhite compositioncontaining triethyl phosphatehaving enhancedwhipping properties.U.S. Patent 3,328,175,June 27. DAWES, D. R., HOLLAND, P., andRUMENS, M. J. (1960).The relationshipbetweenthe chemical structure and neurotoxicity of alkyl organophosphoruscompounds.Brit. J. Pharmacol. 15, 271-278. DUBOIS,K. P., and MANGUN, G. H. (1947). Effect of hexaethyl tetraphosphateon choline esterasein vitro and in vivo. Proc. Sot. Exptl. Biol. Med. 64, 137-139. DUNCAN, D. B. (1955).Multiple rangeand multiple F. tests.Biometrics 11, l-42. FLEISHER, J. H., and POPE, E. J. (1954).Calorimetric method for determinationof red blood cell cholinesterase activity in whole blood. Arch. Znd. Hyg. Occupational Med. 9, 323-334. AMFIROSE,
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FLEISHER,J. H., POPE,E. J., and SPEAR, S. F. (1955). Determination of red blood cell cholinesterase activity in whole blood. Arch. Ind. Health 11, 332-337. KENNEY,F. T. (1959).Propertiesof partially purified tyrosine-a-ketoglutaratetransaminase from rat liver. J. Biol. Chem. 234,2707-2712. KING, E. J., and WOTTON, I. D. P. (1956).Micro-analysis in Medical Biochemistry, 3rd ed., pp. 81-86. Grune & Stratton, New York. MURPHY, S.D. (1965).Mechanismof the effect of acrolein on rat liver enzymes.Toxicol. Appl. Pharmacol.
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MURPHY,S. D.(1966). Responseof adaptive rat liver enzymesto acutepoisoningby organophosphateinsecticides.Toxicol. Appl. Pharmacol. 8,266-276. U.S. Armed ForcesInstitute of Pathology (1960).Manual of Histologic and Special Staining Technics, 2nd ed., pp. 149-150.McGraw-Hill, New York. VANDEKAR,M. (1957).Anesthetic effect producedby organopbosphorus compounds.Nature 179, 154-l 55.