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Potentiation of acrylate ester toxicity by prior treatment with the carboxylesterase inhibitor triorthotolyl phosphate (TOTP)
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
57,
208-219 (1981)
Potentiation of Acrylate Ester Toxicity by Prior Treatment with the Carboxylesterase Inhibitor Triorthotolyl Phosphate (TOTP)‘,” E. H. SILVERY AND S. D. MLJRPHY~ Department
of Physiology,
Kresge Center for Environmental 665 Hungtington Avenue, Boston,
Received
January
Health, Harvard School Massachusetts 02115
30, 1980: accepted
October
of Public
Health.
13. 1980
Potentiation of Acrylate Ester Toxicity by Prior Treatment with the Carboxylesterase Inhibitor Triorthotolyl Phosphate (TOTP). SILVER, E. H., AND MURPHY, S. D. (1981). Toxicol. Appl. Pharmacol. 57, 208-219. The ability of the carboxylesterase inhibitor TOTP to modify the metabolism, mortality, and sulfhydryl depletion resulting from inhalation of acrylate esters was investigated in male rats. Methyl acrylate and ethyl acrylate were enzymatically hydrolyzed by plasma and by homogenates of rat liver, lung, and kidney, with highest activity found in liver homogenates. Hydrolysis of methyl and ethyl acrylate by tissue homogenates from rats treated with TOTP was inhibited in a dose-dependent manner. Pretreatment of rats with 125 mg/kg of TOTP potentiated the lethal action of inhaled methyl acrylate and ethyl acrylate. In the 72-hr period following termination of a 4-hr exposure to 500, 750, or 1000 ppm methyl acrylate the respective mortality rates in TOTP pretreated rats were 83, 100, and 100% compared with 0, 17. and 67% mortality in corn oil-pretreated rats. The acrylate esters reacted with glutathione in vitro and decreased tissue nonprotein sulfhydryl (NPSH) in vivo. The depletion of NPSH by inhaled acrylate esters was most pronounced in lung compared with that in liver, kidney, or blood. TOTP pretreatment significantly enhanced acrylate ester-induced decreases in tissue NPSH concentrations. In rats exposed for 4 hr to 135, 370, 490, or 720 ppm methyl acrylate, lung NPSH was reduced 34, 55, 69, and 83%, respectively, in TOTP-pretreated rats versus 0. 26, 27, and 52%, respectively, in corn oil-pretreated rats. Kidney NPSH following exposure to acrylate esters was significantly altered only in TOTP-pretreated rats. These studies demonstrate that carboxylesterases are important in the detoxification of methyl a&ate and ethyl acrylate and that exposure to inhibitors of carboxylesterases may potentiate the adverse effects of these esters.
Inhibition by TOTP (triorthotolyl phosphate) and EPN (0-ethyl-0-p-nitrophenyl, phenyl phosphonothioate) of tissue carboxylesterases necessary for the hydrolytic detoxification of malathion is a primary factor in their enhancement of malathion’s toxicity (Frawley et al., 1957; Murphy and DuBois, 1957; Murphy et al., 1959; Cohen and Murphy, 1971). Some organophosphates, including EPN and TOTP, inhibit carboxylesterases and carboxylamidases at doses lower than those which inhibit acetylcholinesterase. Many studies 0041-008X/81/020208-12$02.00/0 Copyright Q 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
’ Supported by Research Grants ES-00002, ES-00084, and ES-00045 from the National Institute of Environmental Health Sciences, U.S. Department of Health. Education, and Welfare, and by a grant from Procter and Gamble Company. 2 Presented in part at the seventeenth Annual Meeting of the Society of Toxicology held in San Francisco, California in March 1978. 3 Present address: Department of Pathology. Peter Bent Brigham Hospital. 721 Huntington Avenue. Boston, Massachusetts 02115. J Present address: Division of Toxicology. Department of Pharmacology, Medical School. University of Texas Health Science Center at Houston, P.O. Box 20708, Houston, Texas 77025. 208
TOTP INTERACTION
WITH ACRYLATE
have examined toxic interactions between pesticides which inhibit carboxylesterases and pesticides containing ester or amide groups that may be detoxified by esterases (Abernathy and Casida, 1973; Casida et al., 1963; Cohen et al., 1972; Murphy and Cheever, 1972; Singleton and Murphy, 1973). Interactions between esterase inhibitors and organic esters used as drugs have also been examined (Cohen and Orzech, 1977; Heymann et al., 1969; Kolassa et al., 1977; Satoh and Moroi, 1973). Little information is available concerning the importance of tissue carboxylesterases in the metabolism and modulation of toxicity of the many organic esters used industrially. The hydrolysis of ethyl acetate in vitro by rat blood and the accumulation of ethanol in the blood of rats inhaling high concentrations of ethyl acetate have been demonstrated (Gallagher and Loomis, 1975). Recently, metabolites of methyl methacrylate have been described (Bratt and Hathway, 1977). The conversion of 65% of this ester to COP within 2 hr in viva suggests that hydrolysis of the ester occurs rapidly. Neither of these studies examined the importance of carboxylesterases in modifying the adverse effects of these esters. Silver and Murphy (1978) have described the interaction between TOTP and some hepatotoxic esters of ally1 alcohol. The toxicity of these esters is obviously due to the release of ally1 alcohol following hydrolysis of these esters. Esters of acrylic acid are used extensively in industry in the preparation of emulsion polymers for use in latex paints, lacquers, floor polishes, and sealers. Of these, the low-molecular-weight monomers methyl acrylate and ethyl acrylate are in widest use (Autian, 1975; Anonymous, 1977). The acute and subacute toxicities of inhaled methyl acrylate and ethyl acrylate have been reported (Bezpalko, 1967; Pozzani et al., 1949; Treon et al., 1949). These esters are irritant to mucous membranes and following inhalation the lung is a major site
ESTERS
209
of injury, with changes also reported in liver, kidney, and heart after high concentrations (Pozzani et al., 1949; Treon et al., 1949). The industrial threshold limit values (TLV) established for methyl acrylate and ethyl acrylate are 10 and 25 ppm, respectively. Less information is available concerning the toxic effects of inhaled acrylic acid; however, limited data suggest that the acid may be less acutely toxic than its methyl and ethyl esters (Gage, 1970). Compounds which contain a double bond adjacent to an electron withdrawing group, such as the carbonyl moiety of an ester or aldehyde, are particularly reactive with nucleophiles (Dixon, 1948), and would be expected to react with and deplete nonprotein sulfhydryl. Esters of acrylic acid possess this type of structure. The thiol most abundant in cells is glutathione (GSH). Its cellular concentration is normally at least an order of magnitude greater than that of other nonprotein sulfhydryl compounds (Jocelyn, 1972). The present study was conducted to examine the role of carboxylesterases in the metabolism of the acrylate esters and to investigate the potential for interaction of these esters with a carboxylesterase inhibitor (TOTP). In addition to mortality studies, depletion of nonprotein sulfhydryl (NPSH) was investigated as an index of acrylate ester toxicity and interaction with TOTP. METHODS Male Holtzman rats (160-240 g) were used in these studies. The animals were housed five or six per cage in rooms maintained on a 12-hr light-dark cycle. Food (Purina Rat Chow) and water were available at all times except during exposure periods. Methyl acrylate (99%), ethyl acrylate (98.5%), and acrylic acid (99%) were purchased from Aldrich Chemical Co., Inc., Milwaukee, Wisconsin. These acrylates contained hydroquinone monomethyl ether as a polymerization inhibitor. Methyl acrylate and ethyl acrylate were distilled prior to use. Due to hazards involved in heating acrylic acid, it was used as obtained from the supplier. Triorthotolyl phos-
210
SILVER AND MURPHY
phate (TOTP), practical, was obtained from EastPrior to assay, organs were placed in 0.02 M EDTA and frozen at -30°C. Assays were completed as soon man Organic Chemicals, Rochester, New York. as possible after sacrifice, normally within 3 days. The 5.5’-Dithiobis-(2)nitrobenzoic acid (DTNB) was supstatistical significance of differences between group plied by Sigma Chemical Company, St. Louis, Missouri. means was evaluated using Student’s t test. A value of TOTP was administered 18 hr prior to either sacri- p < 0.05 is considered to indicate a significant difference between group means. Where necessary, Dunfice of rats for determination of tissue esterase activities or the start of an inhalation exposure to nett’s t test for multiple comparisons was used. acrylates. TOTP was dissolved in corn oil in concenThe reactivity of glutathione (GSH) with methyl trations so that the desired dose could be administered acrylate, ethyl acrylate, and acrylic acid was measby the ip injection of 1 ml/kg. Control animals received ured as described by Hashimoto and Aldridge ( 1970). corn oil 1 ml/kg. Rats were sacrificed by cervical tranAn acrylate compound (5 mM) was incubated at 37°C section and exsanguinated. with GSH (5 mM), in 0.1 M phosphate buffer (pH 7.3) Carboxylesterase assays were performed manocontaining 1.5 x lo-* M KCN. At 1 min intervals, a metrically by a modification of the method described sample was removed from the reaction mixture by Murphy (1967) for malathion hydrolysis. Methyl and the amount of GSH remaining in the mixture was acrylate and ethyl acrylate were used as substrates in measured as described above for NPSH. these assays. The substrates, suspended in 0.026 M bicarbonate buffer containing 5% Triton X-100, were added from the sidearm of Warburg flasks to RESULTS homogenates of tissue in bicarbonate buffer. The flasks were equilibrated with shaking at 37°C for 5 Hydrolysis of Methyl and Ethyl Acrylate by min, then the CO, evolved during a 20 min period was measured and the micromoles of acrylic acid Rat Tissues formed were calculated (~1 CO, at STP + 22.4). Substrate concentrations used in these assays were Homogenates of rat tissues were assayed selected on the basis of results of preliminary studies for their ability to hydrolyze methyl and to provide conditions under which tissue concentraethyl acrylate. Assay conditions were setion was the rate-limiting factor in hydrolysis of the esters, that is, at substrate saturation conditions. lected, based on results obtained from preInhalation exposures were conducted under dyliminary studies, so that tissues could be namic conditions in 30-liter chambers originally decompared at approximately equal activity. scribed by Leach (1963) and modified (Conolly er al., Table 1 summarizes the experimental con1978) to permit regulation of the interior temperaditions and control levels of hydrolysis by ture. A variable speed infusion pump (Harvard Apparatus Co.. Model 944) was used to meter methyl plasma and by homogenates of liver, lung, acrylate or ethyl acrylate into dilution air to achieve and kidney. Esterase activity toward ethyl desired concentrations of these esters in the exposure and methyl acrylate was highest in liver. chamber. Atmospheres of acrylic acid were genHomogenates of rat lung were approxierated by passing a metered air stream through acrylic mately one-fourth as active as rat liver. Rat acid and diluting the resulting air mixture to obtain the appropriate concentration. Rats were placed in the kidney was also less active than rat liver. It exposure chambers 15-30 min prior to the start of an was necessary to use a greater quantity of exposure to permit adjustment to the chamber environkidney homogenate and/or higher substrate ment. All exposures lasted 4 hr. concentrations in order to obtain assay acChamber air was sampled every 15 min during extivities equal to liver. The hydrolytic acposure. Methyl acrylate, ethyl acrylate, and acrylic acid concentrations were determined on a Hewletttivity of plasma toward these esters was low. Packard 5720A gas chromatograph equipped with a flame ionization detector, using a 5 ft x ‘/8 in. stainEffect of TOTP Treatment on the Hydrolless-steel Poropak Q column. With an oven temperaysis of Methyl and Ethyl Acrylate ture of 220°C and carrier gas (nitrogen) flow rate of 50 ml/min, the retention times of methyl acrylate, ethyl acrylate, and acrylic acid were 50, 79, and 133 set, TOTP, an inhibitor of carboxylesterases, respectively. was used in these studies to investigate the Whole blood and homogenates of liver, lungs, and effect of inhibition of esterases on the kidneys were assayed for NPSH content according to toxicity of the acrylate esters. An experithe calorimetric method of Sedlak and Lindsay (1968).
TOTP INTERACTION
WITH ACRYLATE TABLE
211
ESTERS
1
HYDROLYSISOFMETHYLACRYLATEANDETHYLACRYLATEBYTISSUEHOMOGENATESFROMCONTROLRATS Substrate
concentration Tissue
Quantity
Substrate
pmol” hydrolyzed
(M)
Liver Liver
40 mg 40 mg
Methyl acrylate Ethyl acrylate
0.042 0.042
8.88 2 0.31 10.40 t 0.40
Lung Lung
150 mg 150 mg
Methyl acrylate Ethyl acrylate
0.042 0.042
9.78 2 0.71 8.71 f 0.45
Kidney Kidney
100 mg 40 mg
Methyl acrylate Ethyl acrylate
0.25 0.25
7.77 2 0.49 8.48 t 0.54
Plasma Plasma
0.5 ml 0.5 ml
Methyl acrylate Ethyl acrylate
0.25 0.25
5.67 + 0.31 6.74 -c 0.63
’ Value is mean + SE pmol of acrylic acid formed per 20 min incubation with tissue homogenates from six rats.
ment was performed to determine the dose-response relationship for TOTP-induced inhibition of methyl and ethyl acrylate hydrolysis. Groups of six rats were given ip injections of corn oil or varying doses of TOTP and sacrificed 18 hr later. Esterase assays on plasma and homogenates of liver, lung, and kidney were per-
formed using the conditions described in Table 1. As shown in Table 2, hydrolysis of both methyl and ethyl acrylate by all tissues examined was markedly and significantly (p < 0.05) inhibited following treatment with 5 mg/kg TOTP. The extent of inhibition increased with increasing doses of TOTP.
TABLE
2
EFFECTOF TOTP ONTHEHYDROLYSISOFMETHYLACRYLATEANDETHYLACRYLATE BYRATTISSUE HOMOGENATES Dose of TOTP (mg/kg)” Tissue
Substrate
5
10
25 (% Inhibition
50
125
of hydrolysis)
Liver Liver
Methyl acrylate Ethyl acrylate
58.2 2 6.8 56.3 k 9.3
63.8 + 13.6 66.2 2 14.0
84.1 2 4.3 85.9 + 4.2
84.5 2 3.6 84.6 lr 3.0
95.5 lr 1.3 91.1 t 2.0
Lung Lung
Methyl acrylate Ethyl acrylate
40.5 + 11.6 39.8 k 9.7
38.4 k 24.6 43.8 k 22.5
76.9 + 5.7 76.7 k 5.9
77.0 f 2.5 74.9 + 4.8
86.6 f 2.9 86.0 -t 3.2
Kidney Kidney
Methyl a&ate Ethyl acrylate
41.7 f 9.0 39.9 ? 11.2
57.5 k 10.3 73.6 k 13.7
77.5 k 2.7 92.3 k 2.6
65.4 + 7.0 89.7 k 2.5
77.0 * 1.9 97.4 + 1.0
Plasma Plasma
Methyl acrylate Ethyl acrylate
42.4 2 12.2 35.5 2 11.3
55.5 ” 10.7 47.2 + 16.7
59.5 2 3.6 83.5 + 4.0
62.3 k 5.3 61.8 f 6.8
75.4 f 2.2 81.4 r 2.2
a Rats, six per group, were pretreated with TOTP or corn oil. Eighteen hours later the animals were sacrificed and homogenates of their tissues were assayed for their capacity to hydrolyze methyl and ethyl acrylate. Values represent mean ? SE percentage inhibition of ester hydrolysis.
212
SILVER
AND TABLE
EFFECT
OF TOTP
PRETREATMENT
MURPHY 3
OF RATS EXPIXED FOR 4 OR ACRYLIC ACID
ON MORTALITY ETHYL ACRYLATE,
hr TO METHYL ACRYLATE.
Pretreatment
_--
Corn oil Exposure Methyl acrylate
Ethyl acrylate
Acrylic acid
Concentration @pm)
Mortality”
200 365 500 750 1000 1500
O/6 O/6 O/6 l/6 416 616
300 500 750 1000 1500
O/6 O/6 O/6 O/6 l/6
1300 1600 2100
O/6 616 515
TOTP
Time of death -
Mortality
Time of death
O/6 016 516 616 616 616
.-.. 8 to 72 hr PE DE to 20 hr PE DE” DE
48-72 hr PE
016 O/6 516 516 616
_ -~ DE to 20 hr PE DE to 20 hr PE DP
DE DE
016 6/6 5/5
DE DE
24-48 hr PE” 8-20 hr PE 8-20 hr PE -
” Observed during the 72-hr period following termination of a 4 hr inhalation exposure; mortality = number died/number tested. * PE, postexposure. r DE, during exposure.
Effect of TOTP Pretreatment on the Acute Toxicity of Methyl Acrylate, Ethyl Acrylate, and Acrylic Acid Results of the esterase assays indicated that methyl acrylate and ethyl acrylate were hydrolyzed by rat tissue esterases and that TOTP inhibited this hydrolysis. A series of experiments were next conducted to investigate the effect of pretreatment with TOTP on the acute toxicity of methyl and ethyl acrylate and acrylic acid. Rats were pretreated with 125 mg/kg TOTP or with corn oil 18 hr prior to the start of an inhalation exposure. The rats were exposed for 4 hr to air containing acrylic acid or an acrylate ester. Exposures were started between 7 and 9 AM. At least five rats from each pretreatment group served as controls and were exposed to room air. During and following the exposures, animals were observed for
adverse effects. Mortality for the 72-hr period following termination of the exposure was recorded and used to compare pretreatment groups. Results of a preliminary experiment indicated that mortality measured at 72 hr did not differ from that at 1 week following exposure to methyl acrylate . Within minutes after initiation of exposure to the higher concentrations of acrylate esters or acrylic acid, rats exhibited signs of nasal and eye irritation. As the exposure continued, the rats developed dyspnea and a scruffy appearance. Convulsions occurred prior to the death of rats exposed to high concentrations of methyl and ethyl acrylate. Mortality data are summarized in Table 3. Methyl acrylate appeared to be the most toxic of the three chemicals. Pretreatment with TOTP, 125 mg/kg, potentiated the acute toxicity of methyl and ethyl acrylate
TOTP INTERACTION TABLE NONPROTEIN
SULFHYDRYL IN TISSUES
WITH ACRYLATE
4 (NPSH)
CONCENTRATION
OF AIR-EXPOSED
RATS
Pretreatment TOTP Tissue Lung Blood Liver Kidney
Corn oil 0.177 0.089 0.623 0.362
lr 0.004” t 0.003 k 0.017 -r- 0.004
(125wi&.d 0.185 0.0% 0.797 0.383
f f f it
0.004 0.003 0.026* 0.004*
a Concentration expressed as mean k SE mmol of NPSH/lOO g wet weight of tissue. Data from eight experiments were pooled. For both corn oil and TOTP pretreatment groups n = 39. * Significantly different from corn oil pretreatment group @ < 0.001).
as indicated both by numbers of deaths and survival times. The potency of methyl acrylate, with respect to lethality, was approximately doubled following TOTP pretreatment. As shown in Table 3, the toxicity of ethyl acrylate was also enhanced following treatment with TOTP. These acrylate esters at higher concentrations killed several TOTP-pretreated rats during the exposure periods. Deaths among corn-oil pretreated rats exposed to similar ester concentrations occurred at later times, never during the exposure period. The toxicity of acrylic acid was not altered by prior administration of TOTP. Effect of TOTP Pretreatment on Acrylate Ester-Induced Depletion of NPSH The reactivities of methyl acrylate, ethyl acrylate, and acrylic acid with GSH were compared at pH 7.3. During a 5-min incubation at 37”C, the decreases in GSH concentration were as follows: methyl acrylate 54%, ethyl acrylate 53%, and acrylic acid 3%. It seemed possible that measurement of tissue NPSH concentration would be a sensitive index of the effect of acrylate esters on various organs and could be used
ESTERS
213
to compare the effects of these esters and acrylic acid on tissues of TOTP and corn oil-pretreated rats. Average tissue NPSH concentrations of air-exposed rats 23 hr following the administration of corn oil or TOTP, 125 mg/kg, are listed in Table 4. Lung and blood NPSH levels were similar in corn oil and TOTPpretreated rats when data from eight experiments involving 39 rats per group were pooled. Kidney NPSH concentrations were slightly (6%) but significantly (p < 0.001) higher in TOTP-pretreated rats compared with corn oil-pretreated rats. TOTP administration, alone, consistently resulted in an increase, averaging 28%, in liver NPSH concentration (p < 0.001). In the graphs and tables which follow, NPSH data are expressed as percentage of daily control to permit comparison between exposures conducted on different days. In a preliminary study, NPSH levels of rat tissues were measured at 1 and 6 hr following termination of a 4 hr exposure to 1000 ppm methyl acrylate. Significant decreases occurred in lung, liver, and blood NPSH. These decreases were more pronounced at 1 hr than at 6 hr postexposure. Based on these data in subsequent experiments rats were sacrificed at 1 hr after termination of exposures to acrylate esters or acrylic acid. Organ to body weight ratios were unaffected by prior TOTP treatment. Exposure to acrylic acid, methyl acrylate, and ethyl acrylate also did not alter organ to body weight ratios in either corn .oil or TOTP-pretreated rats. The results of NPSH assays of rat tissues following exposure to the acrylates are summarized in Figs. 1 to 3. The acrylate esters were more potent than acrylic acid in depleting tissue NPSH. Of the tissues assayed, the lung appeared to be most sensitive to the sulfhydryl depletion caused by inhaled methyl and ethyl acrylate. As shown in Figs. 1 and 2, 370 ppm methyl acrylate and 480 ppm ethyl acrylate decreased lung NPSH 27 and 34%, respectively, in corn oil-
214
SILVER AND MURPHY
loo60
qb.+ CORN OIL
x '....... 1 .....
60 .
=
40 J-h+
2 e z
20- LUNG
“3
'1.. .... T f +. 1 'l__, TOTP
....z h .,,_ ‘i .-....,,
BLOOD
$4............._. Y
-z g
k...'........ ,*.
100 5 g
.‘.__ ‘X0
+-qy 60 60 40.
-\u\ x ..... ..___... # P\ 1 LIVER 200
“'...b...,,, I
'.. &... 600
METHYL
600
1000
ACRYLATE
l.,
'.._ 3
KIDNEY
'..'..‘$ .....______._.. 400
XI
0
200
400
CONCENTRATION
600
800
1000
1 ppm)
FIG. 1. Effect of TOTP pretreatment on methyl acrylate-induced depletion of NPSH. Tissue NPSH concentrations were measured in TOTP or corn oil-pretreated rats following a 4 hr exposure to methyl acrylate. Each point represents the percentage of the respective corn oil or TOTP control concentration based on determination in five animals, except for TOTP pretreated groups exposed to 750 and 1fKKl ppm methyl acrylate where n = 4 and n = 1, respectively, due to deaths which occurred during the exposure. Solid lines refer to corn oil pretreatment while broken lines refer to TOTP pretreatment. # refers to a significant difference (p < 0.05) between means of corn oil and TOTP pretreatment groups.
pretreated rats. Lung NPSH was also significantly decreased in corn oil-pretreated rats following exposure to 1000 ppm acrylic acid. TOTP pretreatment markedly increased the loss of lung NPSH caused by methyl and ethyl acrylate. Lung NPSH was decreased 34% in TOTP-pretreated rats exposed to 130 ppm methyl acrylate as compared with no decrease in corn oil-pretreated rats exposed to this concentration. In these experiments, one of five and four of five rats died during exposure to 750 and 1000 ppm methyl acrylate while four of five rats died during inhalation of 1000 ppm ethyl acrylate. This mortality was consistent with that observed in the acute toxicity studies which demonstrated that TOTP-pretreated rats were more susceptible to the lethal action of high concen-
trations of acrylate esters. The depletion of NPSH caused by acrylic acid was not markedly altered by pretreatment with TOTP. Whole blood NPSH was also significantly lower following exposure to 750 and 1000 ppm methyl acrylate, 1000 ppm ethyl acrylate, and 1000 ppm acrylic acid (Figs. 1, 2, and 3). The decrease in blood NPSH levels was substantially and significantly greater in TOTP-treated rats exposed to either methyl or ethyl acrylate. In TOTPpretreated rats, 290 ppm of ethyl acrylate and 370 ppm of methyl acrylate decreased blood NPSH significantly. The effect of acrylic acid on blood NPSH was not changed by prior treatment with TOTP. The effect of acrylate esters on liver NPSH was variable; however, some significant de-
TOTP INTERACTION
WITH ACRYLATE
215
ESTERS
I00 ::j L f z
b #1
40'
\**
:.,‘.. :..
# I '.. Y., '2.
i$....
$4 . . . . . . . .
20. LUNG 100 :
#>
a...'&..../.."
BLOOD
y&
i,... '...
: '..
E z
60.
+ I '.. '... '... +... .,.\ ... . ..+ #y..,..* 40-t '..* _ LIVER I 260 400 660 600 1000 CO-
ETHYL
ACRYLATE
TOTP \, --.._., x... _............0 i
+
'.. #' .... ‘!t '...._.... .. ......_........ 9 #t
KIDNEY 0
200
CONCENTRATION
460
600
600
1000
(ppml
FIG. 2. Effect of TOTP pretreatment on ethyl acrylate-induced depletion of NPSH. Tissue NPSH concentrations were measured in TOTP or corn oil-pretreated rats following a 4 hr exposure to ethyl acrylate. Each point represents the percentage of the respective corn oil or TOTP control concentration based on determinations in five animals except for the TOTP pretreated group exposed to 1000 ppm ethyl acrylate where n = 1, as a result of deaths among this group of animals during the exposure. Solid lines refer to corn oil pretreatment while broken lines refer to TOTP pretreatment. # refers to a significant difference (p < 0.05) between means of corn oil and TOTP pretreatment.
creases were observed. As in the lung and blood, TOTP pretreatment potentiated the decrease in liver NPSH caused by methyl and ethyl acrylate, but had little effect on acrylic acid induced changes in NPSH. The kidney sustained the least acrylate ester-induced depletion of NPSH (Figs. 1 and 2). Significant changes in renal NPSH were not observed in corn oil-pretreated rats exposed to the acrylate esters. In rats pretreated with TOTP, both methyl and ethyl acrylate caused significant decreases in renal NPSH. Renal NPSH was 50% lower in corn oil-pretreated rats exposed to 1000 ppm acrylic acid as compared to air-exposed controls. TOTP did not affect acrylic acid induced depletion of kidney NPSH. While these results demonstrated that prior treatment with TOTP (125 mg/kg) potentiated acrylate ester induced depletion
of NPSH, results of the initial studies of acrylate ester hydrolysis indicated that 5 mg/kg of TOTP significantly inhibited the hydrolysis of both methyl acrylate and ethyl acrylate. To further relate the enhancement of acrylate ester toxicity to the inhibitory effect of TOTP on ester hydrolysis, a doseresponse study of TOTP’s ability to augment methyl acrylate’s decrease in lung NPSH was conducted. Following pretreatment with corn oil or 5, 10, 25, 50, or 125 mg/kg of TOTP, rats were exposed for 4 hr to 500 ppm of methyl acrylate. This concentration of methyl acrylate was selected because in previous experiments it significantly decreased lung NPSH in corn oilpretreated rats without maximally depleting lung NPSH in TOTP-pretreated rats. As shown in Table 5, lung NPSH content in air exposed rats was not significantly affected
216
SILVER AND MURPHY
~............... * .,,_, “/““OIL Y >
80 2 E0 UP
TOTP
60
-llNG
60
:
‘J
BLOOD
--.... ..X. ‘.., ‘l\ #\\:., \\\ 7>
40
ACRYLIC ACID CONCENTRATION(ppm) FIG. 3. Effect of TOTP pretreatment on acrylic acid-induced depletion of NPSH. Tissue NPSH concentrations were measured in TOTP or corn oil-pretreated rats following 4 hr exposure to acrylic acid. Each point represents the percentage of the respective corn oil or TOTP control concentration based on determinations in five animals except for groups exposed to 1000 ppm where n = 4, due to the deaths of one animal from the corn oil and TOTP pretreatment groups. Solid lines refer to corn oil pretreatment while broken lines refer to TOTP pretreatment. # refers to a significant difference between means of corn oil and TOTP pretreated groups.
18 hr after pretreatment with any dose of TOTP. Lung NPSH levels were reduced in rats of all pretreatment groups following exposure to methyl acrylate. Additionally, the depletion of lung NPSH caused by methyl acrylate was potentiated by TOTP in a dose-dependent manner with significant potentiation occurring after pretreatment with 25, 50, or 125 mg/kg TOTP. DISCUSSION The available data concerning the toxicity of inhaled methyl acrylate, ethyl acrylate, and acrylic acid suggest that the esters may be more toxic than acrylic acid (Gage, 1970; Pozzani et al., Carpenter, 1949; Smyth and Carpenter, 1948; Treon et al., 1949). Additionally, Dixon (1948) has
suggested that sensory irritation and lacrimation may occur as a result of the reaction of chemicals with protein -SH groups of mucous membranes. He notes that compounds, such as ethyl acrylate, which contain a double bond adjacent to the carbonyl group of an ester or aldehyde react with nucleophilic groups such as -SH and are lacrimators. The importance of the double bond of acrylate esters with respect to production of their toxic effects is also suggested by test results indicating that methyl acrylate and ethyl acrylate are approximately lo-fold more toxic (Treon rt al., 1949) and more irritating (Fassett, 1963) than their saturated analogs methyl propionate and ethyl propionate. A rate constant for the reaction of GSH with ethyl acrylate was reported as approximately 600
TOTP INTERACTION TABLE
WITH ACRYLATE
5
EFFECT OF PRETREATMENT WITH VARIOUS DOSES OF TOTP ON METHYL ACRYLATE (500 ppm) INDUCED DEPLETION OF LUNG NPSH
Percentage of control lung NPSH exposure Pretreatment Corn oil 1 ml/kg TOTP 5 ~gflrg 10 mdks 25 m3&3 50 m&3 125 mg/kg
Methyl acrylate (500 mm)
Air 100 94.8 96.8 91.4 96.8 92.3
+ 6.1”
66.5 + 3.9
2 k + k t
61.3 55.5 49.7 41.3 38.1
3.2 3.2 5.2 6.5 2.6
k k + 2 +
4.5 6.1 3.9 3.96 1.9
(1Mean percentage of control k SE for six rats in each group. b Significantly different @ < 0.05) from corn-oilpretreated methyl acrylate exposed group.
times that of GSH with sodium acrylate (Hashimoto and Aldridge, 1970). Our measurements confirmed the greater reactivity of methyl acrylate and ethyl acrylate with GSH at physiologic conditions as compared to that of acrylic acid with GSH. Since acrylate esters are more reactive with nucleophiles than acrylic acid, the enzymatic hydrolysis of the acrylate esters could constitute a detoxification pathway. The results of our experiments indicate that, under substrate saturation conditions, methyl acrylate and ethyl acrylate are hydrolyzed at similar rates by rat plasma, liver, and lung in vitro. However, ethyl acrylate was hydrolyzed three times more rapidly than methyl acrylate by kidney homogenates. Pretreatment of rats with TOTP, an inhibitor of nonspecific esterases, caused a dose-dependent decrease in the hydrolysis of methyl acrylate and ethyl acrylate by their tissues in vitro. Esterase activity in mammals is reported to be highest in liver, kidney, duodenum, and brain (Krisch, 1971). In this study, maximal
ESTERS
217
hydrolysis of acrylate esters occurred in liver homogenates. Lung homogenates were also quite active in hydrolyzing acrylate esters. With histochemical methods, nonspecific esterases have been localized in the epithelial cells extending from the airways down to the alveoli (Sorokin, 1970). It is possible that these lung enzymes, with their relatively high hydrolytic activity toward methyl and ethyl acrylate, and their location in the epithelium, have an important role in the metabolism and detoxification of inhaled acrylate esters. Additional support for a role of esterases in the detoxification of the acrylate esters was provided by results of acute inhalation toxicity studies. Mortalities at 72 hr after a 4-hr inhalation exposure showed that methyl acrylate was more toxic than ethyl acrylate. TOTP (125 mg/kg) pretreatment potentiated the toxicity of both methyl and ethyl acrylate approximately twofold. Mortality following acrylic acid exposure was unaltered by prior TOTP administration. Alterations in tissue NPSH concentration also proved to be a sensitive index for comparison of the effect of acrylate esters on various tissues following TOTP pretreatment. Inhalation exposure to acrylate esters lowered NPSH concentrations, particularly in the lung. At concentrations up to 500 ppm the decrease in lung NPSH was similar for methyl acrylate and ethyl acrylate. Above this concentration, methyl acrylate further depleted lung P-IPSH, whereas the decrease in lung NPSH due to ethyl acrylate reached a plateau. Acrylate ester-induced alterations in liver, kidney, and blood NPSH were less notable. In subacute toxicity studies of ethyl acrylate (Pozzani et al., 1949), major histopathologic changes were found in lungs of exposed animals with less severe changes observed in liver and kidneys of some animals. Thus, our measurements of lowered tissue NPSH concentration as an index of injury from acrylate esters are consistent with reported histopathological observations.
218
SILVER
AND MURPHY
Pretreatment with 12.5 mg/kg TOTP markedly augmented the depletion of NPSH produced by the acrylate esters. Following TOTP pretreatment the acrylate esters caused substantial decreases in liver, blood, and kidney NPSH levels, however, the greatest decreases again occurred in the lung. The degree of enhancement of methyl acrylate-induced depletion of lung NPSH was TOTP dose-dependent with 25 mg/kg producing significant potentiation. In contrast to the results of our in vitro studies which indicated that acrylic acid reacted only slightly with GSH, inhalation of a high concentration (1000 ppm) of acrylic acid markedly reduced liver and kidney NPSH levels. TOTP pretreatment had little effect on changes in tissue NPSH content produced by exposure to acrylic acid. In addition to the direct chemical reaction of ethyl acrylate with GSH, the enzymatic conjugation of these compounds has been demonstrated by Boyland and Chasseaud (1967). These authors have suggested that glutathione transferases involved in the conjugation reaction may be responsible for the detoxification of ethyl acrylate. Although metabolism of acrylates by GSH-S-transferases were not measured in this investigation, a previous study demonstrated that pretreatment of rats with TOTP did not alter the metabolism of paraoxon and methyl paraoxon via aryl and/or alkyl GSH-S-transferases (Benke and Murphy, 1974). While glutathione transferases may be involved in the metabolism and detoxification of acrylate esters, our data suggest that carboxylesterases are also important detoxification pathways for these esters. The results of these studies also suggest that the toxicity of acrylic acid as compared with that of the esters may occur through different mechanisms. If the effects of acrylate esters were produced following hydrolysis to acrylic acid, inhibition of esterase activity would be expected to protect against the acute toxic action of the esters since acrylic acid was less acutely toxic by
inhalation than the methyl and ethyl esters. This was not the case. Further, acrylic acid reduced liver NPSH concentration more than that of other tissues, whereas the acrylate esters primarily lowered lung NPSH concentration. The hydrolytic pathway may be particularly important for acrylate ester detoxification at low exposure concentrations, in that at low concentrations of methyl acrylate and ethyl acrylate, sulfhydryl depletion occurred only in TOTP-pretreated animals. ACKNOWLEDGMENT The assistance of Miss Sally Reed is acknowledged with appreciation.
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C. O.,
AND
CASIDA,
.I. E. (1973).
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throid insecticides: Esterase cleavage in relation to selective toxicity. Science 179, 1235- 1236. ANONYMOUS (1977). Facts and figures for the chemical industry. Chem. Eng. News 55, 39-79. AUTIAN, J. (1975). Structure-toxicity relationships of acrylic monomers. Environ. Health Perspec. 11, 141- 152. BENKE,
G.
of TOTP paraoxon Chem.
M..
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MURPHY,
S. D.
(1974).
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pretreatment on paraoxon and methyl detoxification in rats. Res. C~~mmucn.
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L. E. (1967). The permissible content of methyl acrylate in the atmosphere. Hvs. S~nit. 32, 3-7.
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(1967).
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zyme-catalyzed conjugations of glutathione with unsaturated compounds. Biothem. J. 104, 95- 102. BRATT, H., AND HATHWAY, D. E. (1977). Fate of methyl methacrylate in rats. Brit. .I. Ccrncrr 36, 114-119. CASIDA, J. E.. BARON, R. L., ETO. M., AND EHGLE, J. L. (1%3). Potentiation and neurotoxicity induced by certain organophosphates. Biochem. Phormncol. 12, 73-83. COHEN, S. D., CALLAGHAN, J. E., AND MURPHY, S. D. (1972). Investigation of multiple mechanisms for potentiation of malaoxon’s anticholinesterase action by triorthotolylphosphate. Proc. Sot. Exp. Biol. Med. 141, 906-910. COHEN, S. D., AND MURPHY, S. D. (1971). Carboxylesterase inhibition as an indicator of malathion
TOTP INTERACTION potentiation
in mice. J. Pharmacol. Exp. Thu.
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COHEN, S. D., AND ORZECH, D. (1977). Triorthotolylphosphate inhibition of carboxylesterases and potentiation ofprocaine toxicity. Biochem. Pharmacol. 26, 1791-1795. CONOLLY, R. B., JAEGER, R. J., AND SZABO, S. (1978). Acute hepatotoxicity of ethylene, vinyl fluoride, vinyl chloride, and vinyl bromide after Aroclor 1254 pretreatment. Exp. Mol. Pathol. 28, 25-33.
DIXON, M. (1948). Reaction of lachrymators with enzymes and proteins. Biochem. Sot. Symp. 2, 39-49.
FASSETT, D. W. (1963). Esters. In Industrial Hygiene and Toxicology (D. W. Fassett and D. D. Irish, eds.), Vol. 2, pp. 1847-1934. Interscience, New York. FRAWLEY, J. P., FUYAT, H. N., HAGAN, E. C., BLAKE, J. R., AND FITZHUGH, 0. G. (1957). Marked potentiation in mammalian toxicity from simultaneous administration of two anticholinesterase compounds. J. Pharmncol. Exp. Thu. 121, 96-106. GAGE, J. C. (1970). The subacute inhalation toxicity of 109 industrial chemicals. Brir. J. Znd. Med. 27, l-18. GALLAGHER, E. J., AND LOOMIS, T. A. (1975). Metabolism of ethyl acetate in the rat: Hydrolysis to ethyl alcohol in vitro and in vivo. Toxicol. Appl. Pharmacol.
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HASHIMOTO, K., AND ALDRIDGE, W. N. (1970). Biochemical studies on acrylamide, a neurotoxic agent. Biochem.
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HEYMANN, E., KRISCH, K., BUCH, H., AND BUZELLO, W. (1969). Inhibition of phenacetin- and acetanilideinduced methemoglobinemia in the rat by the carboxylesterase inhibitor bis-(p-nitrophenyl) phosphate. Biochem. Pharmacol. 18,801-811. JOCELYN, P. C. (1972). Biochemistry ofthe SHGroup. Academic Press, London. KOLASSA, N., TUISL, E., AND KRAUPP, 0. (1977). Effects of the carboxylesterase inhibitor bis-(pnitrophenyb-phosphate on disposition and metabolism of hexobendine. Biochem. Pharmacol. 26, 223-228.
KRISCH, K. (1971). Carboxylic ester bydrolases. In The Enzymes (P. D. Boyer, ed.), 3rd ed., Vol. 5, pp. 43-69. Academic Press, New York.
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ESTERS
LEACH, L. J. (1963). A laboratory test chamber for studying airborne materials. USAEC Research and Development Report, UR-629, I- 12. MURPHY, S. D. (1967). Malathion inhibition of esterases as a determinant of malathion toxicity. J. Pharmacol.
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MURPHY, S. D., ANDERSON, R. L., AND DUBOIS, K. P. (1959). Potentiation of the toxicity of malathion by triorthotolyl phosphate. Proc. Exp. Biol. Med. 100,483-487.
M~JRPHY, S. D., and DUBOIS, K. P. (1957). Quantitative measurement of inhibition of the enzymatic detoxification of malathion by EPN (ethyl pnitrophenyl thionobenzenephosphonate). Proc. Sot. Exp. Biol. Med. %, 813-818. MURPHY, S. D., AND CHEEVER, K. L. (1972). Carboxylesterase and cholinesterase inhibition in rats: abate interaction with malathion. Arch. Environ. Health 24, 107- 114. POZZANI, U. L., WEIL, C. S., AND CARPENTER, C. P. (1949). Subacute vapor toxicity and range-finding data for ethyl a&ate. J. Znd. Hyg. Tox. 31, 311-316. SATOH, T., AND MOROI, K. (1973). Comparative studies on the inhibition of liver amidases, amino peptidase, and serum cholinesterase by EPN. Toxicol. Appl.
Pharmacol.
25, 553-559.
SEDLAK, J., AND LINDSAY, R. H. (1968). Estimation of total, protein-bound and nonprotein sulthydryl groups in tissue with Ellman’s reagent. Anal. Biothem. 25, 192-205. SILVER, E. H., AND MURPHY, S. D. (1978). Effect of carboxylesterase inhibitors on the acute hepatotoxicity of esters of ally1 alcohol. Toxicol. Appl. Pharmacol.
45, 377-389.
SINGLETON, S. D., AND MURPHY, S. D. (1973). Propanil (3,4-dichloropropanilide)-induced methemoglobin formation in mice in relation to acylamidase activity. Toxicol. Appl. Pharmacol. 25, 20-29. SMYTH, H. F., AND CARPENTER, C. P. (1948). Further experience with the range-finding test in the industrial toxicology laboratory. J. Znd. Hyg. Toxicol. 30,63-68.
SOROKIN, S. P. (1970). The cells of the lung. In Morphology of Experimental sis. AEC Symp. Ser.
Respiratory
Carcinogene-
21,3. TREON, J. F., SIGMON, H., WRIGHT, H., AND KITZMILLER, K. U. (1949). The toxicity of methyl and ethyl acrylate. .Z. Znd. Hyg. Tox. 31, 317-326.
AND
APPLIED
PHARMACOLOGY
57,
208-219 (1981)
Potentiation of Acrylate Ester Toxicity by Prior Treatment with the Carboxylesterase Inhibitor Triorthotolyl Phosphate (TOTP)‘,” E. H. SILVERY AND S. D. MLJRPHY~ Department
of Physiology,
Kresge Center for Environmental 665 Hungtington Avenue, Boston,
Received
January
Health, Harvard School Massachusetts 02115
30, 1980: accepted
October
of Public
Health.
13. 1980
Potentiation of Acrylate Ester Toxicity by Prior Treatment with the Carboxylesterase Inhibitor Triorthotolyl Phosphate (TOTP). SILVER, E. H., AND MURPHY, S. D. (1981). Toxicol. Appl. Pharmacol. 57, 208-219. The ability of the carboxylesterase inhibitor TOTP to modify the metabolism, mortality, and sulfhydryl depletion resulting from inhalation of acrylate esters was investigated in male rats. Methyl acrylate and ethyl acrylate were enzymatically hydrolyzed by plasma and by homogenates of rat liver, lung, and kidney, with highest activity found in liver homogenates. Hydrolysis of methyl and ethyl acrylate by tissue homogenates from rats treated with TOTP was inhibited in a dose-dependent manner. Pretreatment of rats with 125 mg/kg of TOTP potentiated the lethal action of inhaled methyl acrylate and ethyl acrylate. In the 72-hr period following termination of a 4-hr exposure to 500, 750, or 1000 ppm methyl acrylate the respective mortality rates in TOTP pretreated rats were 83, 100, and 100% compared with 0, 17. and 67% mortality in corn oil-pretreated rats. The acrylate esters reacted with glutathione in vitro and decreased tissue nonprotein sulfhydryl (NPSH) in vivo. The depletion of NPSH by inhaled acrylate esters was most pronounced in lung compared with that in liver, kidney, or blood. TOTP pretreatment significantly enhanced acrylate ester-induced decreases in tissue NPSH concentrations. In rats exposed for 4 hr to 135, 370, 490, or 720 ppm methyl acrylate, lung NPSH was reduced 34, 55, 69, and 83%, respectively, in TOTP-pretreated rats versus 0. 26, 27, and 52%, respectively, in corn oil-pretreated rats. Kidney NPSH following exposure to acrylate esters was significantly altered only in TOTP-pretreated rats. These studies demonstrate that carboxylesterases are important in the detoxification of methyl a&ate and ethyl acrylate and that exposure to inhibitors of carboxylesterases may potentiate the adverse effects of these esters.
Inhibition by TOTP (triorthotolyl phosphate) and EPN (0-ethyl-0-p-nitrophenyl, phenyl phosphonothioate) of tissue carboxylesterases necessary for the hydrolytic detoxification of malathion is a primary factor in their enhancement of malathion’s toxicity (Frawley et al., 1957; Murphy and DuBois, 1957; Murphy et al., 1959; Cohen and Murphy, 1971). Some organophosphates, including EPN and TOTP, inhibit carboxylesterases and carboxylamidases at doses lower than those which inhibit acetylcholinesterase. Many studies 0041-008X/81/020208-12$02.00/0 Copyright Q 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
’ Supported by Research Grants ES-00002, ES-00084, and ES-00045 from the National Institute of Environmental Health Sciences, U.S. Department of Health. Education, and Welfare, and by a grant from Procter and Gamble Company. 2 Presented in part at the seventeenth Annual Meeting of the Society of Toxicology held in San Francisco, California in March 1978. 3 Present address: Department of Pathology. Peter Bent Brigham Hospital. 721 Huntington Avenue. Boston, Massachusetts 02115. J Present address: Division of Toxicology. Department of Pharmacology, Medical School. University of Texas Health Science Center at Houston, P.O. Box 20708, Houston, Texas 77025. 208
TOTP INTERACTION
WITH ACRYLATE
have examined toxic interactions between pesticides which inhibit carboxylesterases and pesticides containing ester or amide groups that may be detoxified by esterases (Abernathy and Casida, 1973; Casida et al., 1963; Cohen et al., 1972; Murphy and Cheever, 1972; Singleton and Murphy, 1973). Interactions between esterase inhibitors and organic esters used as drugs have also been examined (Cohen and Orzech, 1977; Heymann et al., 1969; Kolassa et al., 1977; Satoh and Moroi, 1973). Little information is available concerning the importance of tissue carboxylesterases in the metabolism and modulation of toxicity of the many organic esters used industrially. The hydrolysis of ethyl acetate in vitro by rat blood and the accumulation of ethanol in the blood of rats inhaling high concentrations of ethyl acetate have been demonstrated (Gallagher and Loomis, 1975). Recently, metabolites of methyl methacrylate have been described (Bratt and Hathway, 1977). The conversion of 65% of this ester to COP within 2 hr in viva suggests that hydrolysis of the ester occurs rapidly. Neither of these studies examined the importance of carboxylesterases in modifying the adverse effects of these esters. Silver and Murphy (1978) have described the interaction between TOTP and some hepatotoxic esters of ally1 alcohol. The toxicity of these esters is obviously due to the release of ally1 alcohol following hydrolysis of these esters. Esters of acrylic acid are used extensively in industry in the preparation of emulsion polymers for use in latex paints, lacquers, floor polishes, and sealers. Of these, the low-molecular-weight monomers methyl acrylate and ethyl acrylate are in widest use (Autian, 1975; Anonymous, 1977). The acute and subacute toxicities of inhaled methyl acrylate and ethyl acrylate have been reported (Bezpalko, 1967; Pozzani et al., 1949; Treon et al., 1949). These esters are irritant to mucous membranes and following inhalation the lung is a major site
ESTERS
209
of injury, with changes also reported in liver, kidney, and heart after high concentrations (Pozzani et al., 1949; Treon et al., 1949). The industrial threshold limit values (TLV) established for methyl acrylate and ethyl acrylate are 10 and 25 ppm, respectively. Less information is available concerning the toxic effects of inhaled acrylic acid; however, limited data suggest that the acid may be less acutely toxic than its methyl and ethyl esters (Gage, 1970). Compounds which contain a double bond adjacent to an electron withdrawing group, such as the carbonyl moiety of an ester or aldehyde, are particularly reactive with nucleophiles (Dixon, 1948), and would be expected to react with and deplete nonprotein sulfhydryl. Esters of acrylic acid possess this type of structure. The thiol most abundant in cells is glutathione (GSH). Its cellular concentration is normally at least an order of magnitude greater than that of other nonprotein sulfhydryl compounds (Jocelyn, 1972). The present study was conducted to examine the role of carboxylesterases in the metabolism of the acrylate esters and to investigate the potential for interaction of these esters with a carboxylesterase inhibitor (TOTP). In addition to mortality studies, depletion of nonprotein sulfhydryl (NPSH) was investigated as an index of acrylate ester toxicity and interaction with TOTP. METHODS Male Holtzman rats (160-240 g) were used in these studies. The animals were housed five or six per cage in rooms maintained on a 12-hr light-dark cycle. Food (Purina Rat Chow) and water were available at all times except during exposure periods. Methyl acrylate (99%), ethyl acrylate (98.5%), and acrylic acid (99%) were purchased from Aldrich Chemical Co., Inc., Milwaukee, Wisconsin. These acrylates contained hydroquinone monomethyl ether as a polymerization inhibitor. Methyl acrylate and ethyl acrylate were distilled prior to use. Due to hazards involved in heating acrylic acid, it was used as obtained from the supplier. Triorthotolyl phos-
210
SILVER AND MURPHY
phate (TOTP), practical, was obtained from EastPrior to assay, organs were placed in 0.02 M EDTA and frozen at -30°C. Assays were completed as soon man Organic Chemicals, Rochester, New York. as possible after sacrifice, normally within 3 days. The 5.5’-Dithiobis-(2)nitrobenzoic acid (DTNB) was supstatistical significance of differences between group plied by Sigma Chemical Company, St. Louis, Missouri. means was evaluated using Student’s t test. A value of TOTP was administered 18 hr prior to either sacri- p < 0.05 is considered to indicate a significant difference between group means. Where necessary, Dunfice of rats for determination of tissue esterase activities or the start of an inhalation exposure to nett’s t test for multiple comparisons was used. acrylates. TOTP was dissolved in corn oil in concenThe reactivity of glutathione (GSH) with methyl trations so that the desired dose could be administered acrylate, ethyl acrylate, and acrylic acid was measby the ip injection of 1 ml/kg. Control animals received ured as described by Hashimoto and Aldridge ( 1970). corn oil 1 ml/kg. Rats were sacrificed by cervical tranAn acrylate compound (5 mM) was incubated at 37°C section and exsanguinated. with GSH (5 mM), in 0.1 M phosphate buffer (pH 7.3) Carboxylesterase assays were performed manocontaining 1.5 x lo-* M KCN. At 1 min intervals, a metrically by a modification of the method described sample was removed from the reaction mixture by Murphy (1967) for malathion hydrolysis. Methyl and the amount of GSH remaining in the mixture was acrylate and ethyl acrylate were used as substrates in measured as described above for NPSH. these assays. The substrates, suspended in 0.026 M bicarbonate buffer containing 5% Triton X-100, were added from the sidearm of Warburg flasks to RESULTS homogenates of tissue in bicarbonate buffer. The flasks were equilibrated with shaking at 37°C for 5 Hydrolysis of Methyl and Ethyl Acrylate by min, then the CO, evolved during a 20 min period was measured and the micromoles of acrylic acid Rat Tissues formed were calculated (~1 CO, at STP + 22.4). Substrate concentrations used in these assays were Homogenates of rat tissues were assayed selected on the basis of results of preliminary studies for their ability to hydrolyze methyl and to provide conditions under which tissue concentraethyl acrylate. Assay conditions were setion was the rate-limiting factor in hydrolysis of the esters, that is, at substrate saturation conditions. lected, based on results obtained from preInhalation exposures were conducted under dyliminary studies, so that tissues could be namic conditions in 30-liter chambers originally decompared at approximately equal activity. scribed by Leach (1963) and modified (Conolly er al., Table 1 summarizes the experimental con1978) to permit regulation of the interior temperaditions and control levels of hydrolysis by ture. A variable speed infusion pump (Harvard Apparatus Co.. Model 944) was used to meter methyl plasma and by homogenates of liver, lung, acrylate or ethyl acrylate into dilution air to achieve and kidney. Esterase activity toward ethyl desired concentrations of these esters in the exposure and methyl acrylate was highest in liver. chamber. Atmospheres of acrylic acid were genHomogenates of rat lung were approxierated by passing a metered air stream through acrylic mately one-fourth as active as rat liver. Rat acid and diluting the resulting air mixture to obtain the appropriate concentration. Rats were placed in the kidney was also less active than rat liver. It exposure chambers 15-30 min prior to the start of an was necessary to use a greater quantity of exposure to permit adjustment to the chamber environkidney homogenate and/or higher substrate ment. All exposures lasted 4 hr. concentrations in order to obtain assay acChamber air was sampled every 15 min during extivities equal to liver. The hydrolytic acposure. Methyl acrylate, ethyl acrylate, and acrylic acid concentrations were determined on a Hewletttivity of plasma toward these esters was low. Packard 5720A gas chromatograph equipped with a flame ionization detector, using a 5 ft x ‘/8 in. stainEffect of TOTP Treatment on the Hydrolless-steel Poropak Q column. With an oven temperaysis of Methyl and Ethyl Acrylate ture of 220°C and carrier gas (nitrogen) flow rate of 50 ml/min, the retention times of methyl acrylate, ethyl acrylate, and acrylic acid were 50, 79, and 133 set, TOTP, an inhibitor of carboxylesterases, respectively. was used in these studies to investigate the Whole blood and homogenates of liver, lungs, and effect of inhibition of esterases on the kidneys were assayed for NPSH content according to toxicity of the acrylate esters. An experithe calorimetric method of Sedlak and Lindsay (1968).
TOTP INTERACTION
WITH ACRYLATE TABLE
211
ESTERS
1
HYDROLYSISOFMETHYLACRYLATEANDETHYLACRYLATEBYTISSUEHOMOGENATESFROMCONTROLRATS Substrate
concentration Tissue
Quantity
Substrate
pmol” hydrolyzed
(M)
Liver Liver
40 mg 40 mg
Methyl acrylate Ethyl acrylate
0.042 0.042
8.88 2 0.31 10.40 t 0.40
Lung Lung
150 mg 150 mg
Methyl acrylate Ethyl acrylate
0.042 0.042
9.78 2 0.71 8.71 f 0.45
Kidney Kidney
100 mg 40 mg
Methyl acrylate Ethyl acrylate
0.25 0.25
7.77 2 0.49 8.48 t 0.54
Plasma Plasma
0.5 ml 0.5 ml
Methyl acrylate Ethyl acrylate
0.25 0.25
5.67 + 0.31 6.74 -c 0.63
’ Value is mean + SE pmol of acrylic acid formed per 20 min incubation with tissue homogenates from six rats.
ment was performed to determine the dose-response relationship for TOTP-induced inhibition of methyl and ethyl acrylate hydrolysis. Groups of six rats were given ip injections of corn oil or varying doses of TOTP and sacrificed 18 hr later. Esterase assays on plasma and homogenates of liver, lung, and kidney were per-
formed using the conditions described in Table 1. As shown in Table 2, hydrolysis of both methyl and ethyl acrylate by all tissues examined was markedly and significantly (p < 0.05) inhibited following treatment with 5 mg/kg TOTP. The extent of inhibition increased with increasing doses of TOTP.
TABLE
2
EFFECTOF TOTP ONTHEHYDROLYSISOFMETHYLACRYLATEANDETHYLACRYLATE BYRATTISSUE HOMOGENATES Dose of TOTP (mg/kg)” Tissue
Substrate
5
10
25 (% Inhibition
50
125
of hydrolysis)
Liver Liver
Methyl acrylate Ethyl acrylate
58.2 2 6.8 56.3 k 9.3
63.8 + 13.6 66.2 2 14.0
84.1 2 4.3 85.9 + 4.2
84.5 2 3.6 84.6 lr 3.0
95.5 lr 1.3 91.1 t 2.0
Lung Lung
Methyl acrylate Ethyl acrylate
40.5 + 11.6 39.8 k 9.7
38.4 k 24.6 43.8 k 22.5
76.9 + 5.7 76.7 k 5.9
77.0 f 2.5 74.9 + 4.8
86.6 f 2.9 86.0 -t 3.2
Kidney Kidney
Methyl a&ate Ethyl acrylate
41.7 f 9.0 39.9 ? 11.2
57.5 k 10.3 73.6 k 13.7
77.5 k 2.7 92.3 k 2.6
65.4 + 7.0 89.7 k 2.5
77.0 * 1.9 97.4 + 1.0
Plasma Plasma
Methyl acrylate Ethyl acrylate
42.4 2 12.2 35.5 2 11.3
55.5 ” 10.7 47.2 + 16.7
59.5 2 3.6 83.5 + 4.0
62.3 k 5.3 61.8 f 6.8
75.4 f 2.2 81.4 r 2.2
a Rats, six per group, were pretreated with TOTP or corn oil. Eighteen hours later the animals were sacrificed and homogenates of their tissues were assayed for their capacity to hydrolyze methyl and ethyl acrylate. Values represent mean ? SE percentage inhibition of ester hydrolysis.
212
SILVER
AND TABLE
EFFECT
OF TOTP
PRETREATMENT
MURPHY 3
OF RATS EXPIXED FOR 4 OR ACRYLIC ACID
ON MORTALITY ETHYL ACRYLATE,
hr TO METHYL ACRYLATE.
Pretreatment
_--
Corn oil Exposure Methyl acrylate
Ethyl acrylate
Acrylic acid
Concentration @pm)
Mortality”
200 365 500 750 1000 1500
O/6 O/6 O/6 l/6 416 616
300 500 750 1000 1500
O/6 O/6 O/6 O/6 l/6
1300 1600 2100
O/6 616 515
TOTP
Time of death -
Mortality
Time of death
O/6 016 516 616 616 616
.-.. 8 to 72 hr PE DE to 20 hr PE DE” DE
48-72 hr PE
016 O/6 516 516 616
_ -~ DE to 20 hr PE DE to 20 hr PE DP
DE DE
016 6/6 5/5
DE DE
24-48 hr PE” 8-20 hr PE 8-20 hr PE -
” Observed during the 72-hr period following termination of a 4 hr inhalation exposure; mortality = number died/number tested. * PE, postexposure. r DE, during exposure.
Effect of TOTP Pretreatment on the Acute Toxicity of Methyl Acrylate, Ethyl Acrylate, and Acrylic Acid Results of the esterase assays indicated that methyl acrylate and ethyl acrylate were hydrolyzed by rat tissue esterases and that TOTP inhibited this hydrolysis. A series of experiments were next conducted to investigate the effect of pretreatment with TOTP on the acute toxicity of methyl and ethyl acrylate and acrylic acid. Rats were pretreated with 125 mg/kg TOTP or with corn oil 18 hr prior to the start of an inhalation exposure. The rats were exposed for 4 hr to air containing acrylic acid or an acrylate ester. Exposures were started between 7 and 9 AM. At least five rats from each pretreatment group served as controls and were exposed to room air. During and following the exposures, animals were observed for
adverse effects. Mortality for the 72-hr period following termination of the exposure was recorded and used to compare pretreatment groups. Results of a preliminary experiment indicated that mortality measured at 72 hr did not differ from that at 1 week following exposure to methyl acrylate . Within minutes after initiation of exposure to the higher concentrations of acrylate esters or acrylic acid, rats exhibited signs of nasal and eye irritation. As the exposure continued, the rats developed dyspnea and a scruffy appearance. Convulsions occurred prior to the death of rats exposed to high concentrations of methyl and ethyl acrylate. Mortality data are summarized in Table 3. Methyl acrylate appeared to be the most toxic of the three chemicals. Pretreatment with TOTP, 125 mg/kg, potentiated the acute toxicity of methyl and ethyl acrylate
TOTP INTERACTION TABLE NONPROTEIN
SULFHYDRYL IN TISSUES
WITH ACRYLATE
4 (NPSH)
CONCENTRATION
OF AIR-EXPOSED
RATS
Pretreatment TOTP Tissue Lung Blood Liver Kidney
Corn oil 0.177 0.089 0.623 0.362
lr 0.004” t 0.003 k 0.017 -r- 0.004
(125wi&.d 0.185 0.0% 0.797 0.383
f f f it
0.004 0.003 0.026* 0.004*
a Concentration expressed as mean k SE mmol of NPSH/lOO g wet weight of tissue. Data from eight experiments were pooled. For both corn oil and TOTP pretreatment groups n = 39. * Significantly different from corn oil pretreatment group @ < 0.001).
as indicated both by numbers of deaths and survival times. The potency of methyl acrylate, with respect to lethality, was approximately doubled following TOTP pretreatment. As shown in Table 3, the toxicity of ethyl acrylate was also enhanced following treatment with TOTP. These acrylate esters at higher concentrations killed several TOTP-pretreated rats during the exposure periods. Deaths among corn-oil pretreated rats exposed to similar ester concentrations occurred at later times, never during the exposure period. The toxicity of acrylic acid was not altered by prior administration of TOTP. Effect of TOTP Pretreatment on Acrylate Ester-Induced Depletion of NPSH The reactivities of methyl acrylate, ethyl acrylate, and acrylic acid with GSH were compared at pH 7.3. During a 5-min incubation at 37”C, the decreases in GSH concentration were as follows: methyl acrylate 54%, ethyl acrylate 53%, and acrylic acid 3%. It seemed possible that measurement of tissue NPSH concentration would be a sensitive index of the effect of acrylate esters on various organs and could be used
ESTERS
213
to compare the effects of these esters and acrylic acid on tissues of TOTP and corn oil-pretreated rats. Average tissue NPSH concentrations of air-exposed rats 23 hr following the administration of corn oil or TOTP, 125 mg/kg, are listed in Table 4. Lung and blood NPSH levels were similar in corn oil and TOTPpretreated rats when data from eight experiments involving 39 rats per group were pooled. Kidney NPSH concentrations were slightly (6%) but significantly (p < 0.001) higher in TOTP-pretreated rats compared with corn oil-pretreated rats. TOTP administration, alone, consistently resulted in an increase, averaging 28%, in liver NPSH concentration (p < 0.001). In the graphs and tables which follow, NPSH data are expressed as percentage of daily control to permit comparison between exposures conducted on different days. In a preliminary study, NPSH levels of rat tissues were measured at 1 and 6 hr following termination of a 4 hr exposure to 1000 ppm methyl acrylate. Significant decreases occurred in lung, liver, and blood NPSH. These decreases were more pronounced at 1 hr than at 6 hr postexposure. Based on these data in subsequent experiments rats were sacrificed at 1 hr after termination of exposures to acrylate esters or acrylic acid. Organ to body weight ratios were unaffected by prior TOTP treatment. Exposure to acrylic acid, methyl acrylate, and ethyl acrylate also did not alter organ to body weight ratios in either corn .oil or TOTP-pretreated rats. The results of NPSH assays of rat tissues following exposure to the acrylates are summarized in Figs. 1 to 3. The acrylate esters were more potent than acrylic acid in depleting tissue NPSH. Of the tissues assayed, the lung appeared to be most sensitive to the sulfhydryl depletion caused by inhaled methyl and ethyl acrylate. As shown in Figs. 1 and 2, 370 ppm methyl acrylate and 480 ppm ethyl acrylate decreased lung NPSH 27 and 34%, respectively, in corn oil-
214
SILVER AND MURPHY
loo60
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60 .
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ACRYLATE
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400
CONCENTRATION
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800
1000
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FIG. 1. Effect of TOTP pretreatment on methyl acrylate-induced depletion of NPSH. Tissue NPSH concentrations were measured in TOTP or corn oil-pretreated rats following a 4 hr exposure to methyl acrylate. Each point represents the percentage of the respective corn oil or TOTP control concentration based on determination in five animals, except for TOTP pretreated groups exposed to 750 and 1fKKl ppm methyl acrylate where n = 4 and n = 1, respectively, due to deaths which occurred during the exposure. Solid lines refer to corn oil pretreatment while broken lines refer to TOTP pretreatment. # refers to a significant difference (p < 0.05) between means of corn oil and TOTP pretreatment groups.
pretreated rats. Lung NPSH was also significantly decreased in corn oil-pretreated rats following exposure to 1000 ppm acrylic acid. TOTP pretreatment markedly increased the loss of lung NPSH caused by methyl and ethyl acrylate. Lung NPSH was decreased 34% in TOTP-pretreated rats exposed to 130 ppm methyl acrylate as compared with no decrease in corn oil-pretreated rats exposed to this concentration. In these experiments, one of five and four of five rats died during exposure to 750 and 1000 ppm methyl acrylate while four of five rats died during inhalation of 1000 ppm ethyl acrylate. This mortality was consistent with that observed in the acute toxicity studies which demonstrated that TOTP-pretreated rats were more susceptible to the lethal action of high concen-
trations of acrylate esters. The depletion of NPSH caused by acrylic acid was not markedly altered by pretreatment with TOTP. Whole blood NPSH was also significantly lower following exposure to 750 and 1000 ppm methyl acrylate, 1000 ppm ethyl acrylate, and 1000 ppm acrylic acid (Figs. 1, 2, and 3). The decrease in blood NPSH levels was substantially and significantly greater in TOTP-treated rats exposed to either methyl or ethyl acrylate. In TOTPpretreated rats, 290 ppm of ethyl acrylate and 370 ppm of methyl acrylate decreased blood NPSH significantly. The effect of acrylic acid on blood NPSH was not changed by prior treatment with TOTP. The effect of acrylate esters on liver NPSH was variable; however, some significant de-
TOTP INTERACTION
WITH ACRYLATE
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ESTERS
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FIG. 2. Effect of TOTP pretreatment on ethyl acrylate-induced depletion of NPSH. Tissue NPSH concentrations were measured in TOTP or corn oil-pretreated rats following a 4 hr exposure to ethyl acrylate. Each point represents the percentage of the respective corn oil or TOTP control concentration based on determinations in five animals except for the TOTP pretreated group exposed to 1000 ppm ethyl acrylate where n = 1, as a result of deaths among this group of animals during the exposure. Solid lines refer to corn oil pretreatment while broken lines refer to TOTP pretreatment. # refers to a significant difference (p < 0.05) between means of corn oil and TOTP pretreatment.
creases were observed. As in the lung and blood, TOTP pretreatment potentiated the decrease in liver NPSH caused by methyl and ethyl acrylate, but had little effect on acrylic acid induced changes in NPSH. The kidney sustained the least acrylate ester-induced depletion of NPSH (Figs. 1 and 2). Significant changes in renal NPSH were not observed in corn oil-pretreated rats exposed to the acrylate esters. In rats pretreated with TOTP, both methyl and ethyl acrylate caused significant decreases in renal NPSH. Renal NPSH was 50% lower in corn oil-pretreated rats exposed to 1000 ppm acrylic acid as compared to air-exposed controls. TOTP did not affect acrylic acid induced depletion of kidney NPSH. While these results demonstrated that prior treatment with TOTP (125 mg/kg) potentiated acrylate ester induced depletion
of NPSH, results of the initial studies of acrylate ester hydrolysis indicated that 5 mg/kg of TOTP significantly inhibited the hydrolysis of both methyl acrylate and ethyl acrylate. To further relate the enhancement of acrylate ester toxicity to the inhibitory effect of TOTP on ester hydrolysis, a doseresponse study of TOTP’s ability to augment methyl acrylate’s decrease in lung NPSH was conducted. Following pretreatment with corn oil or 5, 10, 25, 50, or 125 mg/kg of TOTP, rats were exposed for 4 hr to 500 ppm of methyl acrylate. This concentration of methyl acrylate was selected because in previous experiments it significantly decreased lung NPSH in corn oilpretreated rats without maximally depleting lung NPSH in TOTP-pretreated rats. As shown in Table 5, lung NPSH content in air exposed rats was not significantly affected
216
SILVER AND MURPHY
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ACRYLIC ACID CONCENTRATION(ppm) FIG. 3. Effect of TOTP pretreatment on acrylic acid-induced depletion of NPSH. Tissue NPSH concentrations were measured in TOTP or corn oil-pretreated rats following 4 hr exposure to acrylic acid. Each point represents the percentage of the respective corn oil or TOTP control concentration based on determinations in five animals except for groups exposed to 1000 ppm where n = 4, due to the deaths of one animal from the corn oil and TOTP pretreatment groups. Solid lines refer to corn oil pretreatment while broken lines refer to TOTP pretreatment. # refers to a significant difference between means of corn oil and TOTP pretreated groups.
18 hr after pretreatment with any dose of TOTP. Lung NPSH levels were reduced in rats of all pretreatment groups following exposure to methyl acrylate. Additionally, the depletion of lung NPSH caused by methyl acrylate was potentiated by TOTP in a dose-dependent manner with significant potentiation occurring after pretreatment with 25, 50, or 125 mg/kg TOTP. DISCUSSION The available data concerning the toxicity of inhaled methyl acrylate, ethyl acrylate, and acrylic acid suggest that the esters may be more toxic than acrylic acid (Gage, 1970; Pozzani et al., Carpenter, 1949; Smyth and Carpenter, 1948; Treon et al., 1949). Additionally, Dixon (1948) has
suggested that sensory irritation and lacrimation may occur as a result of the reaction of chemicals with protein -SH groups of mucous membranes. He notes that compounds, such as ethyl acrylate, which contain a double bond adjacent to the carbonyl group of an ester or aldehyde react with nucleophilic groups such as -SH and are lacrimators. The importance of the double bond of acrylate esters with respect to production of their toxic effects is also suggested by test results indicating that methyl acrylate and ethyl acrylate are approximately lo-fold more toxic (Treon rt al., 1949) and more irritating (Fassett, 1963) than their saturated analogs methyl propionate and ethyl propionate. A rate constant for the reaction of GSH with ethyl acrylate was reported as approximately 600
TOTP INTERACTION TABLE
WITH ACRYLATE
5
EFFECT OF PRETREATMENT WITH VARIOUS DOSES OF TOTP ON METHYL ACRYLATE (500 ppm) INDUCED DEPLETION OF LUNG NPSH
Percentage of control lung NPSH exposure Pretreatment Corn oil 1 ml/kg TOTP 5 ~gflrg 10 mdks 25 m3&3 50 m&3 125 mg/kg
Methyl acrylate (500 mm)
Air 100 94.8 96.8 91.4 96.8 92.3
+ 6.1”
66.5 + 3.9
2 k + k t
61.3 55.5 49.7 41.3 38.1
3.2 3.2 5.2 6.5 2.6
k k + 2 +
4.5 6.1 3.9 3.96 1.9
(1Mean percentage of control k SE for six rats in each group. b Significantly different @ < 0.05) from corn-oilpretreated methyl acrylate exposed group.
times that of GSH with sodium acrylate (Hashimoto and Aldridge, 1970). Our measurements confirmed the greater reactivity of methyl acrylate and ethyl acrylate with GSH at physiologic conditions as compared to that of acrylic acid with GSH. Since acrylate esters are more reactive with nucleophiles than acrylic acid, the enzymatic hydrolysis of the acrylate esters could constitute a detoxification pathway. The results of our experiments indicate that, under substrate saturation conditions, methyl acrylate and ethyl acrylate are hydrolyzed at similar rates by rat plasma, liver, and lung in vitro. However, ethyl acrylate was hydrolyzed three times more rapidly than methyl acrylate by kidney homogenates. Pretreatment of rats with TOTP, an inhibitor of nonspecific esterases, caused a dose-dependent decrease in the hydrolysis of methyl acrylate and ethyl acrylate by their tissues in vitro. Esterase activity in mammals is reported to be highest in liver, kidney, duodenum, and brain (Krisch, 1971). In this study, maximal
ESTERS
217
hydrolysis of acrylate esters occurred in liver homogenates. Lung homogenates were also quite active in hydrolyzing acrylate esters. With histochemical methods, nonspecific esterases have been localized in the epithelial cells extending from the airways down to the alveoli (Sorokin, 1970). It is possible that these lung enzymes, with their relatively high hydrolytic activity toward methyl and ethyl acrylate, and their location in the epithelium, have an important role in the metabolism and detoxification of inhaled acrylate esters. Additional support for a role of esterases in the detoxification of the acrylate esters was provided by results of acute inhalation toxicity studies. Mortalities at 72 hr after a 4-hr inhalation exposure showed that methyl acrylate was more toxic than ethyl acrylate. TOTP (125 mg/kg) pretreatment potentiated the toxicity of both methyl and ethyl acrylate approximately twofold. Mortality following acrylic acid exposure was unaltered by prior TOTP administration. Alterations in tissue NPSH concentration also proved to be a sensitive index for comparison of the effect of acrylate esters on various tissues following TOTP pretreatment. Inhalation exposure to acrylate esters lowered NPSH concentrations, particularly in the lung. At concentrations up to 500 ppm the decrease in lung NPSH was similar for methyl acrylate and ethyl acrylate. Above this concentration, methyl acrylate further depleted lung P-IPSH, whereas the decrease in lung NPSH due to ethyl acrylate reached a plateau. Acrylate ester-induced alterations in liver, kidney, and blood NPSH were less notable. In subacute toxicity studies of ethyl acrylate (Pozzani et al., 1949), major histopathologic changes were found in lungs of exposed animals with less severe changes observed in liver and kidneys of some animals. Thus, our measurements of lowered tissue NPSH concentration as an index of injury from acrylate esters are consistent with reported histopathological observations.
218
SILVER
AND MURPHY
Pretreatment with 12.5 mg/kg TOTP markedly augmented the depletion of NPSH produced by the acrylate esters. Following TOTP pretreatment the acrylate esters caused substantial decreases in liver, blood, and kidney NPSH levels, however, the greatest decreases again occurred in the lung. The degree of enhancement of methyl acrylate-induced depletion of lung NPSH was TOTP dose-dependent with 25 mg/kg producing significant potentiation. In contrast to the results of our in vitro studies which indicated that acrylic acid reacted only slightly with GSH, inhalation of a high concentration (1000 ppm) of acrylic acid markedly reduced liver and kidney NPSH levels. TOTP pretreatment had little effect on changes in tissue NPSH content produced by exposure to acrylic acid. In addition to the direct chemical reaction of ethyl acrylate with GSH, the enzymatic conjugation of these compounds has been demonstrated by Boyland and Chasseaud (1967). These authors have suggested that glutathione transferases involved in the conjugation reaction may be responsible for the detoxification of ethyl acrylate. Although metabolism of acrylates by GSH-S-transferases were not measured in this investigation, a previous study demonstrated that pretreatment of rats with TOTP did not alter the metabolism of paraoxon and methyl paraoxon via aryl and/or alkyl GSH-S-transferases (Benke and Murphy, 1974). While glutathione transferases may be involved in the metabolism and detoxification of acrylate esters, our data suggest that carboxylesterases are also important detoxification pathways for these esters. The results of these studies also suggest that the toxicity of acrylic acid as compared with that of the esters may occur through different mechanisms. If the effects of acrylate esters were produced following hydrolysis to acrylic acid, inhibition of esterase activity would be expected to protect against the acute toxic action of the esters since acrylic acid was less acutely toxic by
inhalation than the methyl and ethyl esters. This was not the case. Further, acrylic acid reduced liver NPSH concentration more than that of other tissues, whereas the acrylate esters primarily lowered lung NPSH concentration. The hydrolytic pathway may be particularly important for acrylate ester detoxification at low exposure concentrations, in that at low concentrations of methyl acrylate and ethyl acrylate, sulfhydryl depletion occurred only in TOTP-pretreated animals. ACKNOWLEDGMENT The assistance of Miss Sally Reed is acknowledged with appreciation.
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AND
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COHEN, S. D., AND ORZECH, D. (1977). Triorthotolylphosphate inhibition of carboxylesterases and potentiation ofprocaine toxicity. Biochem. Pharmacol. 26, 1791-1795. CONOLLY, R. B., JAEGER, R. J., AND SZABO, S. (1978). Acute hepatotoxicity of ethylene, vinyl fluoride, vinyl chloride, and vinyl bromide after Aroclor 1254 pretreatment. Exp. Mol. Pathol. 28, 25-33.
DIXON, M. (1948). Reaction of lachrymators with enzymes and proteins. Biochem. Sot. Symp. 2, 39-49.
FASSETT, D. W. (1963). Esters. In Industrial Hygiene and Toxicology (D. W. Fassett and D. D. Irish, eds.), Vol. 2, pp. 1847-1934. Interscience, New York. FRAWLEY, J. P., FUYAT, H. N., HAGAN, E. C., BLAKE, J. R., AND FITZHUGH, 0. G. (1957). Marked potentiation in mammalian toxicity from simultaneous administration of two anticholinesterase compounds. J. Pharmncol. Exp. Thu. 121, 96-106. GAGE, J. C. (1970). The subacute inhalation toxicity of 109 industrial chemicals. Brir. J. Znd. Med. 27, l-18. GALLAGHER, E. J., AND LOOMIS, T. A. (1975). Metabolism of ethyl acetate in the rat: Hydrolysis to ethyl alcohol in vitro and in vivo. Toxicol. Appl. Pharmacol.
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KRISCH, K. (1971). Carboxylic ester bydrolases. In The Enzymes (P. D. Boyer, ed.), 3rd ed., Vol. 5, pp. 43-69. Academic Press, New York.
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ESTERS
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M~JRPHY, S. D., and DUBOIS, K. P. (1957). Quantitative measurement of inhibition of the enzymatic detoxification of malathion by EPN (ethyl pnitrophenyl thionobenzenephosphonate). Proc. Sot. Exp. Biol. Med. %, 813-818. MURPHY, S. D., AND CHEEVER, K. L. (1972). Carboxylesterase and cholinesterase inhibition in rats: abate interaction with malathion. Arch. Environ. Health 24, 107- 114. POZZANI, U. L., WEIL, C. S., AND CARPENTER, C. P. (1949). Subacute vapor toxicity and range-finding data for ethyl a&ate. J. Znd. Hyg. Tox. 31, 311-316. SATOH, T., AND MOROI, K. (1973). Comparative studies on the inhibition of liver amidases, amino peptidase, and serum cholinesterase by EPN. Toxicol. Appl.
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SEDLAK, J., AND LINDSAY, R. H. (1968). Estimation of total, protein-bound and nonprotein sulthydryl groups in tissue with Ellman’s reagent. Anal. Biothem. 25, 192-205. SILVER, E. H., AND MURPHY, S. D. (1978). Effect of carboxylesterase inhibitors on the acute hepatotoxicity of esters of ally1 alcohol. Toxicol. Appl. Pharmacol.
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