TOXICOLOGY
AND
APPLIED
A Proposed
PHARMACOLOGY
Mechanism
35,481-490
(1976)
for Trimethylphosphate-Induced Sterility1e2
R. D. HARBISON,C. DWIVEDI AND M. A. EVANS Department Department
Received
of Pharmacology and Center in Toxicology, of Biochemistry, Vanderbilt Medical Center, Nashville, Tennessee 37232 July
15, 1975;
accepted
September
18, 1975
A Proposed Mechanism for Trimethylphosphate-Induced Sterility. R. D., DWIVEDI, C. AND EVANS, M. A. (1976). Toxicol. Appl. Pharmacol. 35, 481-490. Trimethylphosphate(TMP) induced sterility in three species,mice.rats, andrabbits. TMP-induced sterility wasdependent on doseand duration of treatment. Sterility was produced within 1 to 2 weeksfollowing initiation of treatment and in all caseswas reversible. Sterility could be maintained by weekly treatment. Fertility returned to normal within 1 to 2 weeksafter termination of treatment. TMP primarily affectsepididymal spermatozoa,probably by an action on spermmotility. TMP suppressed spermatozoacholine acetyltransferaseactivity. Suppression of choline acetyltransferaseactivity wascorrelatedwith TMP-induced sterility. A developmentalpattern of cholineacetyltransferasewasobserved in epididymal spermatozoa.Choline acetyltransferasewas measuredin humanspermatozoa,and valuescomparedto thoseof rat andrabbit sperm. Spermatozoaof all three speciescontainednascentacetylcholineand were capableof synthesizingand releasingacetylcholine in vitro. Inhibition of spermatozoacholine acetyltransferaseactivity impairsspermatozoafunction and producessterility. HARBISON,
Trimethylphosphate (TMP) is used as a gasoline additive, at a concentration of approximately 250 mg per gallon, for controlling surface ignition and spark plug fouling (Hinkamps and Warren, 1968). TMP is also used as a methylating agent (Billman et al., 1942), an intermediate for production of polymethyl polyphosphates (Toy, 1949), a flame retardant solvent for paints and polymers, and a catalyst in preparation of polymers and resins (Hinkamps and Warren, 1968). TMP has also been proposed as a food additive for stabilizing eggwhites (Gumbmann et al., 1968). Tri-alkyl phosphatesare reactive towards nucleophiles and have beenusedto alkylate a variety of functional groups (Jones, 1970).The di-alkyl phosphate moiety is an integral part of a number of organophosphorus pesticides.Recently, TMP has been reported to possessa functional sterilizing action in male rodents (Jackson and Jones, 1968).These investigators described the antifertility action of TMP in rodents and concluded that five successivedosesof TMP produced sterility by action on post-meiotic cells without effects on sperm motility but with a delayed action on other spermatogenic stages. 1This researchwassupportedby NIH grants,No. ES00267and ESO0782. M. A. Evans was supportedby Grant No. GM 00058. z A preliminary report of this work was presented at the fall meeting of the American Society for Pharmacology and Experimental Therapeutics, Universite de Montrkal, MontrCal, Queb& Canada. Copyright Q 1976 by Academic Press, Inc. 481 All rights of reproduction in any form reserved. Printed
in Great
Britain
482
HARBISON,
DWIVEDI
AND
EVANS
TMP is weakly toxic to rodents (Deichmann and Witherup, 1946). Long-term administration, 6 to 79 days, of high dosages, 400 mg/kg to 2 g/kg per day, to rabbits produced weight loss and a functional muscle paralysis. TMP-induced paralysis is reversible, with signs that suggest an effect of TMP on the cholinergic system of muscle. The compound is rapidly degraded to dimethyl phosphate, and S-methyl cysteine has been identified as a urinary metabolite (Jackson and Jones, 1968). The metabolism of organophosphorus pesticides and tri-aryl phosphates is well documented, but little is known about the fate and actions of the simple tri-alkyl phosphates. Even though these compounds are suspected of reacting chemically by an alkylating mechanism, their effects on specific biochemical systems have not been described. The purpose of this investigation was to determine the mechanism of TMP-induced sterility. Because of an apparent effect of TMP on the cholinergic system of skeletal muscle, our study was concentrated on the cholinergic system of the spermatozoa as the possible locus of TMP-induced sterility. METHODS Trimethylphosphate (TMP) (97 %, Aldrich Chemical Co., Milwaukee, Wisconsin) was injected ip or administered by gavage. Random-bred albino Sprague-Dawley-descendant rats (Harlan Industries. Inc., Cumberland, Indiana), random-bred albino Swiss-origin mice (Harlan Industries, Inc., Cumberland, Indiana), or New Zealand white rabbits (Hilltop Rabbit Ranch, Columbia, Tennessee) of proven fertility were used for breeding. Fecundity was measured by serial mating of male mice and rats with demonstrated fertility to corresponding females. One male was placed in a cage with two or three females for I -week duration. At the end of this time, females were separated and new females introduced into the cage at weekly intervals. Females were sacrificed on Day 14 of gestation or on the 14th day mid-week of the mating period. Determination of the state of pregnancy was made at that time. Male rabbits of proven fertility were placed in a cage with a doe in heat and allowed to mate once. Fecundity in all cases was calculated by determining the number of pregnancies per total breeding population. Spermatozoa were sampled from various segments of the epididymis. The caput, proximal corpus, distal corpus, proximal cauda, or distal cauda was minced in Eagle’s medium, and the total number of spermatozoa was determined. Fresh human sperm was obtained by ejaculation. After liquifaction, it was diluted with Hanks’ solution and centrifuged. This process was repeated twice to wash the spermatozoa. Finally, spermatozoa were resuspended in Hanks’ solution and counted. These sperm suspensions were used for measurement of choline acetyltransferase. Choline acetyltransferase activity was assayed by measurement of the formation of 14C-labeled acetylcholine from choline and “C-labeled acetyl-coenzyme A (New England Nuclear, Boston, Massachusetts). This procedure was modified from that reported by McCaman and Hunt (1965). One milliliter of each sperm suspension was homogenized with 4 ml of phosphate buffer (pH 7.4,0.5 M) containing NaCN (0.02 M) and EDTA (0.005 M), and 0.1 ml of the homogenate was used for incubation. One-tenth milliliter of buffer substrate reagent containing phosphate buffer, NaCl, MgC12, physostigmine sulfate, labeled and unlabeled acetyl-coenzyme A and choline was preincubated with 0.1 ml of
TMP-INDUCED
STERILITY
483
phosphate buffer (0.05 M, pH 7.4) for 5 min at 37°C; 0.1 ml of sperm homogenate in phosphate buffer was then added and further incubated for 10 min at 37°C. The final volume of 0.3 ml contained 0.05 M KH,PO, buffer, 0.3 M NaCl, 0.02 M MgCl,, 0.0002 M physostigmine sulfate, 0.006 M NaCN, 0.0016 M EDTA, 5 x low4 M choline iodide, and 5 x lo-’ M total acetyl-coenzyme A. The reaction was stopped by placing the tubes in an ice bath, and 0.1 ml of incubation mixture was passed through a column of anionexchange resin (Bio-Rad AG 1-X8). The column was then washed with 2 ml of distilled water in successive 4 x 0.5-ml portions. Eluate was collected in a scintillation vial to which 15 ml of Aquasol (New England Nuclear) was added and then counted for 14C in a Packard Tri-Carb Model 3320 liquid scintillation counter. Specific activity was calculated to determine acetylcholine production. No choline acetyltransferase activity was detectable in samples prepared as described above which were free of spermatozoa. Thus, choline acetyltransferase activity is located in the spermatozoa. Acetylcholine in sperm was assayed by pyrolysis gas chromatography (Harbison et al., 1975). Spermatozoa were suspended in Norman Johnson solution and then homogenized in 2 % trichloroacetic acid in acetonitrile and centrifuged. The resulting supernatant was extracted twice with equal volumes of ether. The ether layer was discarded and residual ether completely removed under a stream of nitrogen. A 3-ml aliquot of this solution was mixed with 3 ml of dipicyrlamine in dichloromethane (1 mM) and 1.5 ml of NaHCO, buffer (1.75 M, pH 10.7) and shaken for 2 min. The upper aqueous phase was discarded, and to the lower organic phase 3 ml of 0.1 N HCl were added and the mixture shaken 1 min. A 2.5 ml aliquot of the upper phase was transferred to a centrifuge tube and mixed with trimethyl ammonium iodide (0.2 ml, 200 pg/ml) and I,-KI solution (0.2 ml, 10 g of KI and 9 g of I, per 50 ml of distilled water). This mixture was allowed to stand in ice for 30 min, centrifuged at O”C, and blown to dryness under nitrogen. Each centrifuge tube was washed with 50 ~1 of acetonitrile, and the washing was transferred to a platinum ribbon for acetylcholine determination using a HewlettPackard Model 5750 gas chromatograph coupled with a Nuclear Chicago 5180 pyrolyzer. The column temperature was 140°C and the detector temperature was 160°C. Propionyl choline (Mann Research Labs, Inc., New York, New York) (5 nmol) was added before ether extraction as an internal standard. Extracts of human spermatozoa were treated with butyryl chloride (Aldrich Chemical Co., Milwaukee, Wisconsin) to esterify the excessive amounts of choline which interfere with the measurement of acetylcholine. Esterification eliminates this problem. The authenticity of acetylcholine from tissue was confirmed by mass spectrometry. Statistical comparisons between sample means were made by the two-tailed grouped Student’s t test. The level of significance chosen in all cases was p < 0.05. RESULTS Trimethylphosphate (TMP) induced reversible sterility in male mice (Fig. 1). TMPinduced sterility was dependent on both dosage as well as duration of treatment. TMP, 750 mg/kg, administered for 5 consecutive days, produced 13 “/, fecundity during the first week following termination of treatment. However, TMP, 1.5 g/kg, produced total sterility during the first week following termination of treatment and 29 % fecundity
484
HARBISON,
DWIVEDI
AND
EVANS
during the second week. Both of these treatment schedules produced reversible sterility, and, at 1 to 2 weeks following treatment, fertility was normal. The greatest effect on fertility was produced by TMP at 1.5 g/kg administered 5 days/week for 1 month. Following termination of treatment, sterility persisted for 2 weeks, and fertility gradually returned to normal 6 weeks following treatment. Similarly, in male rats, TMP induced reversible sterility that was dependent on both dosage and duration of treatment (Fig. 2). TMP, 100 mg/kg administered for 5 consecutive days/week for 1 month, reduced fecundity to 29 % during the first week following termination of treatment. Fertility returned to normal during the second week. TMP,
: 0’ D *-.-.-
I
*’ 2
3
4
5
6
WEEK
FIG. 1. Fecundity measured by serial mating of male mice. Fecundity in all cases was calculated by determining the number of pregnancies per total breeding population. Trimethyl phosphate was administered for 5 consecutive days and fecundity subsequently measured. Subchronic treatment was 5 days/week for 1 month, and fecundity was subsequently determined. Control animals received the same volume of vehicle.
600 mg/kg consecutively for 5 days, reduced fecundity to O-5% for a 4-week period following termination of the treatment. Fertility returned to normal during the sixth week following treatment. The onset and maintenance of TMP-induced sterility was shown by treatment with TMP, 750 mg/kg, every 7 days. During week 1, fecundity was reduced by 50 %, and, by week 3 and through week 12, fecundity was reduced to O-6 %. TMP also induced sterility in male rabbits which was dependent on both dosage and duration of treatment (Fig. 3). TMP, 200 mg/kg, administered every 5 days reduced fecundity to 50 % by the third week of treatment and to about 25 % by the ninth week. At 325 mg/kg, TMP reduced fecundity to about 37 % by the second week of treatment and produced sterility from week 5 through 13. Fertility returned to normal within 1 week of termination of treatment on week 13. A single treatment of male rabbits with TMP, 750 mg/kg, reduced fecundity to 34 % during the first week following treatment, and fertility was normal during the second week. Mating behavior was similar in both the treated and control groups. Vaginal plugs and mountings were observed with the same frequency in females caged with males in the treated and control groups. Testicular biopsies were taken at various periods during
TMP-INDUCED
485
STERILITY
f-1 ,*’ ,- . .
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WEEK
2. Fecundity measured by serial mating of male rats. Fecundity in all cases was calculated by determining the number of pregnancies per total breeding population. Trimethylphosphate was administered for 5 consecutive days and fecundity subsequently measured. Subchronic treatment was 5 days/week for 1 month, and fecundity was subsequently determined. Chronic treatment was once weekly. Control animals received the same volume of vehicle. FIG.
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FIG. 3. Fecundity measured by placing male rabbits of proven fertility in a cage with a doe in heat and allowing to mate once; 24 hr later, does were sacrified, and ova were flushed from oviducts and examined for evidence of fertilization by determining the presence of pronuclei and cleavage. Fecundity was calculated by determining the number of pregnancies per total breeding population. Trimethylphosphate was administered once weekly and terminated on week 13. Control animals received the same volume of vehicle.
and following treatment. There were no observable histological changes, and spermatogenesis was normal. TMP did not induce observable changes in skeletal muscle activity or animal behavior at the dosages studied.
486
HARBISON,
DWIVEDI
AND
EVANS
Choline acetyltransferase activity of rat spermatozoa was measured (Fig. 4). A developmental pattern was observed. Lowest activity was measured in the caput, 18 pmol of acetylchohne (ACh) synthesized per 1 x 10’ spermatozoa per hour. Tntermediate activity was measured in the corpus, and highest activity was measured in the cauda, 78 pmol of ACh synthesized per 1 x lo5 spermatozoa per hour. TMP treatment, 750 mg/kg, significantly reduced spermatozoa choline acetyltransferase activity from all segments of the epididymis, and continued treatment maintained suppression of this enzymic activity (Fig. 4).
CAPUT
CORPUS
CAUOA
FIG. 4. Acetylcholine synthesis in rat spermatozoa, values represent the mean (+SE) in picomoles of acetylcholine synthesized per IO’ rat spermatozoa per hour. Trimethylphosphate, 750 mg/kg, was administered once weekly for 12 weeks, and enzyme activity was measured 1 week following termination of treatment. Control animals received the same volume of vehicle. Shaded areas, TMP; white areas, control.
The onset and duration of suppressionof choline acetyltransferase activity by TMP is shown in Fig. 5. Maximum suppressionof enzyme activity was observed at 72 hr following treatment, and enzyme activity began to return toward control at 96 to 168hr. A comparison of TMP-induced suppressionof choline acetyltransferase activity was made between the brain enzyme and the spermatozoon enzyme. Spermatozoa choline acetyltransferase was reduced about 50-75 % by TMP while the brain enzyme activity was reduced only about 35% by TMP. Significant depressionof enzyme activity was observed in both spermatozoa as well as brain tissue 24 hr following TMP treatment. The rapid onset of action to depressenzyme activity corresponds to the rapid onset of antifertility activity.
TMP-INDUCED
487
STERILITY
Choline acetyltransferase activity of rabbit spermatozoa was measured (Fig. 6). A developmental pattern was observed. Lowest activity was measured in the caput, 8.0 pmol of acetylcholine (ACh) synthesized per 1 x lo5 spermatozoa per hour, with SPERMATOZOA
‘5
HOURS
POST
TREATMENT
FIG. 5. Acetylcholine synthesis in rat spermatozoa. Values represent the mean (*SE) picomoles of acetylcholine synthesized per lo5 rat spermatozoa per hour. Trimethylphosphate, 750 mg/kg, was administered and animals sacrificed at the specified time periods.
PROXIMAL CORPUS
DISTAL CORPUS
PROXIUAL CAUDA
DISTAL CAUOA
Fm. 6. Acetylcholine synthesis in rabbit spermatozoa. Values represent the mean (&SE) picomoles of acetylcholine synthesized per 10’ rabbit spermatozoa per hour. Trimethylphosphate, 750 mg/kg, was administered once and choline acetyltransferase measured 1 week following treatment. Trimethylphosphate, 200 and 325 mg/kg, was administered weekly for 12 weeks and choline acetyltransferase measured during that week of treatment. Post-treatment measurements of choline acetyltransferase were at 1 to 2 weeks following termination of treatment. Control animals received the same volume of vehicle.
increasing amounts measured in proximal and distal corpus and proximal cauda. Highest levels, six times greater than caput, were measured in the distal cauda, 35 pmol of ACh synthesized per 1 x lo5 spermatozoa per hour. A single TMP treatment reduced spermatozoa choline acetyltransferase activity from all segments of the epididymis, but significant differences were observed only in spermatozoa from proximal cauda and
488
HARBISON.
DWIVEDI
AND
EVANS
distal cauda. Continued weekly treatment with TMP maintained depression of choline acetyltransferase activity. A dose vs response depression of choline acetyltransferase activity was observed when a treatment of TMP of 325 mg/kg was compared to a dosage of 200 mg/kg. A dosage of 325 mg/kg in general produced a greater depression of enzyme activity. Continued TMP treatment at the lower dosages produced as great or greater reductions in choline acetyltransferase activity. TMP-induced reduction of this enzyme system was completely reversible. One to two weeks following termination of treatment, choline acetyltransferase activity returned to normal in spermatozoa from all segments of the epididymis (Fig. 6).
1L iBElT
AN
7. Acetylcholine synthesis in spermatozoa obtained from rats, rabbits, and humans. Values represent the mean (*SE) picomoles of acetylcholine synthesized per 10’ spermatozoa per hour. Rat and rabbit spermatozoa were obtained from the distal cauda; human spermatozoa were obtained fresh by ejaculation. FIG.
Spermatozoa choline acetyltransferase activity was measured in rat, rabbit, and man (Fig. 7). Human spermatozoa choline acetyltransferase activity was assayed to determine ifthis enzyme activity existed in human spermatozoa and to assessthe comparative activities of this sperm enzyme in the human and in laboratory animals. Rat spermatozoa contained the highest enzyme activity. Rabbit and human choline acetyltransferase activities were comparable, about one-half that of the rat. Spermatozoa of all three species contained nascent acetylcholine and were capable of synthesizing and releasing acetylcholine in vitro (Table 1). Human spermatozoa contained the highest amount of nascent ACh but not the highest choline acetyltransferase activity. However, human spermatozoa were capable of synthesizing and releasing, during a 30-min incubation, three to four times the quantity of ACh produced by rat and rabbit sperm.
TMP-INDUCED
489
STERILITY
TABLE 1 SPERMATOZOA ACETYLCHOLINE CONTENT IN SEVERAL SPECIES" Picomoles Species Rabbit Rat Man
Not incubated 18.5 t- 1.1 15.4 &- 1.2 30.6 + 1.9
of ACh/106
spermatozoa
15 minh
30 min
45 min
60 min
20.2 & 1.3 18.6 f 2.2 -
22.2 f 1.2 22.7 -t 2.1 79.3 + 7.1
25.0 + 0.7 32.7 + 2.1 -
31.1 + 0.3 41.1 It. 3.3 -
a Values are means k SE. Not incubated spermatozoa acetylcholine content represent nascent concentrations of acetylcholine. Incubation of spermatozoa with phospholine iodide (1 x low3 M) reflects synthesis and release of acetylcholine into the incubation medium. b Incubation time.
A concentration-dependent inhibition of spermatozoa motility was seen when TMP was added to a sperm suspension in vitro. TMP, 2.5 x lo-’ M, suppressed spermatozoa motility by about 21%. Also in vitro 1 x 10e5 M TMP, inhibited choline acetyltransferase purified from human placenta by about 32%. This was the maximal inhibition that could be obtained in vitro.
DISCUSSION
TMP induces reversible sterility in three species, mouse, rat, and rabbit. Sterility was dependent on dose and duration of treatment and was produced within 1 to 2 weeks following initiation of treatment and in all cases was reversible. Fertility returned to normal within 1 to 2 weeks after termination of treatment. Spermatogenesis was not affected by TMP treatment. TMP primarily affects epididymal spermatozoa, probably by an action on sperm motility. Rabbit spermatozoa were markedly depressed in motility after administration of TMP, and this depression was accompanied by a depression of choline acetyltransferase activity. Mammalian spermatozoa contain high acetylcholinesterase activity, which is concentrated in the flagella (Sekine, 1951; Nelson, 1964, 1966). Spermatozoa preferentially hydrolyze acetylcholine over butyryl- and benzoylcholine (Appelgate and Nelson, 1962; Nelson, 1964). Nelson has proposed a working hypothesis that the acetylcholinesterase may serve to regulate sperm motility by controlling intracellular concentrations of acetylcholine, The synthesis of acetylcholine is controlled by the enzyme choline acetyltransferase. The regulation of sperm motility by acetylcholine is therefore probably dependent on both the rate of acetylcholine synthesis by choline acetyltransferase and the rate of acetylcholine hydrolysis by acetylcholinesterase. These two enzymic actions will control the intracellular content of acetylcholine. Therefore, inhibition of choline acet)rltransferase activity should result in a drop in acetylcholine content and a resultant impairment of spermatozoa motility and ability to fertilize. From the present results, the mechanism of TMP-induced sterility appears to involve inhibition of the spermatozoa enzyme choline acetyltransferase. Inhibition of this enzyme compromises spermatozoa motility and ability to fertilize.
490
HARBISON.
DWIVEDI
AND
EVANS
TMP as well as other tri-alkyl phosphates may inhibit the spermatozoa enzyme choline acetyltransferase and thereby produce a functional sterility. The di-alkyl phosphates useful as organophosphorus pesticides may likewise produce a functional sterility because of their suspected inhibition of choline acetyltransferase. Such a possibility must be experimentally proven in each case. Choline acetyltransferase is present in the sperm of all three species studied, including man. This enzyme appears to be critical for the fertilizing capacity of spermatozoa. Temporary sterility of males exposed to occupational or environmental chemicals may possibly be explained by their effect on the spermatozoa enzyme choline acetyltransferase. The ability of the spermatozoa cholinergic system to regulate its fertilizing capability is a provocative finding. Development of tri-alkyl phosphates or di-alkyl phosphates as potent choline acetyltransferase inhibitors and potential male sterilants is suggested. REFERENCES APPLEGATE,
A.
AND
NELSON,
L. (1962). Acetylcholinesterase in mytilus spermatozoa. Biol.
Bull. 1X3,475-476.
J. H., RADIKE, A. AND MUNDY, B. W. (1942).J. Amer. Chem. Sot. 64,2977-2978. W. B. AND WITHERUP, S. (1946).Observation on effectsof trimethyl phosphate upon experimentalanimals.J. Pharmacol. Exp. Ther. 88, 338-342. GUMBMANN, M. R., GAGNE, W. E. AND WILLIAMS, S. W. (1968).Short-term toxicity studiesof rats fed triethyl phosphatein the diet. Toxicol. Appl. Pharmacol. 12, 360-371. HARBISON, R. D., OLUBADEWO, J., DWIVEDI, C. AND SASTRY, B. V. (1975).Proposedrole of the placentalcholinergicsystemin the regulationof fetal growth and development.In Basic and Therapeutic Aspects of Perinatal Pharmacology (P. L. Morselli, S. Garattini, and F. Sereni,Eds.). Raven Press,New York. HINKAMPS, J. B. AND WARREN, J. A. (1968).Ethyl Ignition Control Compound 4. A Gasoline Additive,for Control of Spark Plug Fouling, Surface Ignition and Rumble. Ethyl Corporation Technical Publication ICC 4. JACKSON, H. AND JONES, A. R. (1968).Antifertility action and metabolismof trimethylphosphate in rodents. Nature (London) 22, 591-592. JONES, A. R. (1970). The metabolismof tri-alkyl phosphates.Experientia 26, 492493. MCCAMAN, R. E., AND HUNT, J. M. (1965). Microdetermination of choline acetylasein nervoustissue.J. Neurochem. 12, 253-259. NELSON, L. (1964).Acetylcholinesterasein bull spermatozoa.J. Reprod. Fert. 7, 65-71. NELSON, L. (1966). Enzyme distribution in “naturally-decapitated” bull spermatozoa: Acetylcholinesterase, adenylpyrophosphataseand adenosinetriphosphatase.J. Cell. Physiol. 68, 113-l 16. SEKINE, T. (1951).Cholinesterase in pig spermatozoa.J. Biochem. (Tokyo) 38,171-179. TOY, A. D. R. (1949). Preparation of tetramethyl pyrophosphate.J. Amer. Chem. Sot. 71, 2268-2269. BILLMAN, DEICHMANN,
AND
APPLIED
A Proposed
PHARMACOLOGY
Mechanism
35,481-490
(1976)
for Trimethylphosphate-Induced Sterility1e2
R. D. HARBISON,C. DWIVEDI AND M. A. EVANS Department Department
Received
of Pharmacology and Center in Toxicology, of Biochemistry, Vanderbilt Medical Center, Nashville, Tennessee 37232 July
15, 1975;
accepted
September
18, 1975
A Proposed Mechanism for Trimethylphosphate-Induced Sterility. R. D., DWIVEDI, C. AND EVANS, M. A. (1976). Toxicol. Appl. Pharmacol. 35, 481-490. Trimethylphosphate(TMP) induced sterility in three species,mice.rats, andrabbits. TMP-induced sterility wasdependent on doseand duration of treatment. Sterility was produced within 1 to 2 weeksfollowing initiation of treatment and in all caseswas reversible. Sterility could be maintained by weekly treatment. Fertility returned to normal within 1 to 2 weeksafter termination of treatment. TMP primarily affectsepididymal spermatozoa,probably by an action on spermmotility. TMP suppressed spermatozoacholine acetyltransferaseactivity. Suppression of choline acetyltransferaseactivity wascorrelatedwith TMP-induced sterility. A developmentalpattern of cholineacetyltransferasewasobserved in epididymal spermatozoa.Choline acetyltransferasewas measuredin humanspermatozoa,and valuescomparedto thoseof rat andrabbit sperm. Spermatozoaof all three speciescontainednascentacetylcholineand were capableof synthesizingand releasingacetylcholine in vitro. Inhibition of spermatozoacholine acetyltransferaseactivity impairsspermatozoafunction and producessterility. HARBISON,
Trimethylphosphate (TMP) is used as a gasoline additive, at a concentration of approximately 250 mg per gallon, for controlling surface ignition and spark plug fouling (Hinkamps and Warren, 1968). TMP is also used as a methylating agent (Billman et al., 1942), an intermediate for production of polymethyl polyphosphates (Toy, 1949), a flame retardant solvent for paints and polymers, and a catalyst in preparation of polymers and resins (Hinkamps and Warren, 1968). TMP has also been proposed as a food additive for stabilizing eggwhites (Gumbmann et al., 1968). Tri-alkyl phosphatesare reactive towards nucleophiles and have beenusedto alkylate a variety of functional groups (Jones, 1970).The di-alkyl phosphate moiety is an integral part of a number of organophosphorus pesticides.Recently, TMP has been reported to possessa functional sterilizing action in male rodents (Jackson and Jones, 1968).These investigators described the antifertility action of TMP in rodents and concluded that five successivedosesof TMP produced sterility by action on post-meiotic cells without effects on sperm motility but with a delayed action on other spermatogenic stages. 1This researchwassupportedby NIH grants,No. ES00267and ESO0782. M. A. Evans was supportedby Grant No. GM 00058. z A preliminary report of this work was presented at the fall meeting of the American Society for Pharmacology and Experimental Therapeutics, Universite de Montrkal, MontrCal, Queb& Canada. Copyright Q 1976 by Academic Press, Inc. 481 All rights of reproduction in any form reserved. Printed
in Great
Britain
482
HARBISON,
DWIVEDI
AND
EVANS
TMP is weakly toxic to rodents (Deichmann and Witherup, 1946). Long-term administration, 6 to 79 days, of high dosages, 400 mg/kg to 2 g/kg per day, to rabbits produced weight loss and a functional muscle paralysis. TMP-induced paralysis is reversible, with signs that suggest an effect of TMP on the cholinergic system of muscle. The compound is rapidly degraded to dimethyl phosphate, and S-methyl cysteine has been identified as a urinary metabolite (Jackson and Jones, 1968). The metabolism of organophosphorus pesticides and tri-aryl phosphates is well documented, but little is known about the fate and actions of the simple tri-alkyl phosphates. Even though these compounds are suspected of reacting chemically by an alkylating mechanism, their effects on specific biochemical systems have not been described. The purpose of this investigation was to determine the mechanism of TMP-induced sterility. Because of an apparent effect of TMP on the cholinergic system of skeletal muscle, our study was concentrated on the cholinergic system of the spermatozoa as the possible locus of TMP-induced sterility. METHODS Trimethylphosphate (TMP) (97 %, Aldrich Chemical Co., Milwaukee, Wisconsin) was injected ip or administered by gavage. Random-bred albino Sprague-Dawley-descendant rats (Harlan Industries. Inc., Cumberland, Indiana), random-bred albino Swiss-origin mice (Harlan Industries, Inc., Cumberland, Indiana), or New Zealand white rabbits (Hilltop Rabbit Ranch, Columbia, Tennessee) of proven fertility were used for breeding. Fecundity was measured by serial mating of male mice and rats with demonstrated fertility to corresponding females. One male was placed in a cage with two or three females for I -week duration. At the end of this time, females were separated and new females introduced into the cage at weekly intervals. Females were sacrificed on Day 14 of gestation or on the 14th day mid-week of the mating period. Determination of the state of pregnancy was made at that time. Male rabbits of proven fertility were placed in a cage with a doe in heat and allowed to mate once. Fecundity in all cases was calculated by determining the number of pregnancies per total breeding population. Spermatozoa were sampled from various segments of the epididymis. The caput, proximal corpus, distal corpus, proximal cauda, or distal cauda was minced in Eagle’s medium, and the total number of spermatozoa was determined. Fresh human sperm was obtained by ejaculation. After liquifaction, it was diluted with Hanks’ solution and centrifuged. This process was repeated twice to wash the spermatozoa. Finally, spermatozoa were resuspended in Hanks’ solution and counted. These sperm suspensions were used for measurement of choline acetyltransferase. Choline acetyltransferase activity was assayed by measurement of the formation of 14C-labeled acetylcholine from choline and “C-labeled acetyl-coenzyme A (New England Nuclear, Boston, Massachusetts). This procedure was modified from that reported by McCaman and Hunt (1965). One milliliter of each sperm suspension was homogenized with 4 ml of phosphate buffer (pH 7.4,0.5 M) containing NaCN (0.02 M) and EDTA (0.005 M), and 0.1 ml of the homogenate was used for incubation. One-tenth milliliter of buffer substrate reagent containing phosphate buffer, NaCl, MgC12, physostigmine sulfate, labeled and unlabeled acetyl-coenzyme A and choline was preincubated with 0.1 ml of
TMP-INDUCED
STERILITY
483
phosphate buffer (0.05 M, pH 7.4) for 5 min at 37°C; 0.1 ml of sperm homogenate in phosphate buffer was then added and further incubated for 10 min at 37°C. The final volume of 0.3 ml contained 0.05 M KH,PO, buffer, 0.3 M NaCl, 0.02 M MgCl,, 0.0002 M physostigmine sulfate, 0.006 M NaCN, 0.0016 M EDTA, 5 x low4 M choline iodide, and 5 x lo-’ M total acetyl-coenzyme A. The reaction was stopped by placing the tubes in an ice bath, and 0.1 ml of incubation mixture was passed through a column of anionexchange resin (Bio-Rad AG 1-X8). The column was then washed with 2 ml of distilled water in successive 4 x 0.5-ml portions. Eluate was collected in a scintillation vial to which 15 ml of Aquasol (New England Nuclear) was added and then counted for 14C in a Packard Tri-Carb Model 3320 liquid scintillation counter. Specific activity was calculated to determine acetylcholine production. No choline acetyltransferase activity was detectable in samples prepared as described above which were free of spermatozoa. Thus, choline acetyltransferase activity is located in the spermatozoa. Acetylcholine in sperm was assayed by pyrolysis gas chromatography (Harbison et al., 1975). Spermatozoa were suspended in Norman Johnson solution and then homogenized in 2 % trichloroacetic acid in acetonitrile and centrifuged. The resulting supernatant was extracted twice with equal volumes of ether. The ether layer was discarded and residual ether completely removed under a stream of nitrogen. A 3-ml aliquot of this solution was mixed with 3 ml of dipicyrlamine in dichloromethane (1 mM) and 1.5 ml of NaHCO, buffer (1.75 M, pH 10.7) and shaken for 2 min. The upper aqueous phase was discarded, and to the lower organic phase 3 ml of 0.1 N HCl were added and the mixture shaken 1 min. A 2.5 ml aliquot of the upper phase was transferred to a centrifuge tube and mixed with trimethyl ammonium iodide (0.2 ml, 200 pg/ml) and I,-KI solution (0.2 ml, 10 g of KI and 9 g of I, per 50 ml of distilled water). This mixture was allowed to stand in ice for 30 min, centrifuged at O”C, and blown to dryness under nitrogen. Each centrifuge tube was washed with 50 ~1 of acetonitrile, and the washing was transferred to a platinum ribbon for acetylcholine determination using a HewlettPackard Model 5750 gas chromatograph coupled with a Nuclear Chicago 5180 pyrolyzer. The column temperature was 140°C and the detector temperature was 160°C. Propionyl choline (Mann Research Labs, Inc., New York, New York) (5 nmol) was added before ether extraction as an internal standard. Extracts of human spermatozoa were treated with butyryl chloride (Aldrich Chemical Co., Milwaukee, Wisconsin) to esterify the excessive amounts of choline which interfere with the measurement of acetylcholine. Esterification eliminates this problem. The authenticity of acetylcholine from tissue was confirmed by mass spectrometry. Statistical comparisons between sample means were made by the two-tailed grouped Student’s t test. The level of significance chosen in all cases was p < 0.05. RESULTS Trimethylphosphate (TMP) induced reversible sterility in male mice (Fig. 1). TMPinduced sterility was dependent on both dosage as well as duration of treatment. TMP, 750 mg/kg, administered for 5 consecutive days, produced 13 “/, fecundity during the first week following termination of treatment. However, TMP, 1.5 g/kg, produced total sterility during the first week following termination of treatment and 29 % fecundity
484
HARBISON,
DWIVEDI
AND
EVANS
during the second week. Both of these treatment schedules produced reversible sterility, and, at 1 to 2 weeks following treatment, fertility was normal. The greatest effect on fertility was produced by TMP at 1.5 g/kg administered 5 days/week for 1 month. Following termination of treatment, sterility persisted for 2 weeks, and fertility gradually returned to normal 6 weeks following treatment. Similarly, in male rats, TMP induced reversible sterility that was dependent on both dosage and duration of treatment (Fig. 2). TMP, 100 mg/kg administered for 5 consecutive days/week for 1 month, reduced fecundity to 29 % during the first week following termination of treatment. Fertility returned to normal during the second week. TMP,
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FIG. 1. Fecundity measured by serial mating of male mice. Fecundity in all cases was calculated by determining the number of pregnancies per total breeding population. Trimethyl phosphate was administered for 5 consecutive days and fecundity subsequently measured. Subchronic treatment was 5 days/week for 1 month, and fecundity was subsequently determined. Control animals received the same volume of vehicle.
600 mg/kg consecutively for 5 days, reduced fecundity to O-5% for a 4-week period following termination of the treatment. Fertility returned to normal during the sixth week following treatment. The onset and maintenance of TMP-induced sterility was shown by treatment with TMP, 750 mg/kg, every 7 days. During week 1, fecundity was reduced by 50 %, and, by week 3 and through week 12, fecundity was reduced to O-6 %. TMP also induced sterility in male rabbits which was dependent on both dosage and duration of treatment (Fig. 3). TMP, 200 mg/kg, administered every 5 days reduced fecundity to 50 % by the third week of treatment and to about 25 % by the ninth week. At 325 mg/kg, TMP reduced fecundity to about 37 % by the second week of treatment and produced sterility from week 5 through 13. Fertility returned to normal within 1 week of termination of treatment on week 13. A single treatment of male rabbits with TMP, 750 mg/kg, reduced fecundity to 34 % during the first week following treatment, and fertility was normal during the second week. Mating behavior was similar in both the treated and control groups. Vaginal plugs and mountings were observed with the same frequency in females caged with males in the treated and control groups. Testicular biopsies were taken at various periods during
TMP-INDUCED
485
STERILITY
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2. Fecundity measured by serial mating of male rats. Fecundity in all cases was calculated by determining the number of pregnancies per total breeding population. Trimethylphosphate was administered for 5 consecutive days and fecundity subsequently measured. Subchronic treatment was 5 days/week for 1 month, and fecundity was subsequently determined. Chronic treatment was once weekly. Control animals received the same volume of vehicle. FIG.
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FIG. 3. Fecundity measured by placing male rabbits of proven fertility in a cage with a doe in heat and allowing to mate once; 24 hr later, does were sacrified, and ova were flushed from oviducts and examined for evidence of fertilization by determining the presence of pronuclei and cleavage. Fecundity was calculated by determining the number of pregnancies per total breeding population. Trimethylphosphate was administered once weekly and terminated on week 13. Control animals received the same volume of vehicle.
and following treatment. There were no observable histological changes, and spermatogenesis was normal. TMP did not induce observable changes in skeletal muscle activity or animal behavior at the dosages studied.
486
HARBISON,
DWIVEDI
AND
EVANS
Choline acetyltransferase activity of rat spermatozoa was measured (Fig. 4). A developmental pattern was observed. Lowest activity was measured in the caput, 18 pmol of acetylchohne (ACh) synthesized per 1 x 10’ spermatozoa per hour. Tntermediate activity was measured in the corpus, and highest activity was measured in the cauda, 78 pmol of ACh synthesized per 1 x lo5 spermatozoa per hour. TMP treatment, 750 mg/kg, significantly reduced spermatozoa choline acetyltransferase activity from all segments of the epididymis, and continued treatment maintained suppression of this enzymic activity (Fig. 4).
CAPUT
CORPUS
CAUOA
FIG. 4. Acetylcholine synthesis in rat spermatozoa, values represent the mean (+SE) in picomoles of acetylcholine synthesized per IO’ rat spermatozoa per hour. Trimethylphosphate, 750 mg/kg, was administered once weekly for 12 weeks, and enzyme activity was measured 1 week following termination of treatment. Control animals received the same volume of vehicle. Shaded areas, TMP; white areas, control.
The onset and duration of suppressionof choline acetyltransferase activity by TMP is shown in Fig. 5. Maximum suppressionof enzyme activity was observed at 72 hr following treatment, and enzyme activity began to return toward control at 96 to 168hr. A comparison of TMP-induced suppressionof choline acetyltransferase activity was made between the brain enzyme and the spermatozoon enzyme. Spermatozoa choline acetyltransferase was reduced about 50-75 % by TMP while the brain enzyme activity was reduced only about 35% by TMP. Significant depressionof enzyme activity was observed in both spermatozoa as well as brain tissue 24 hr following TMP treatment. The rapid onset of action to depressenzyme activity corresponds to the rapid onset of antifertility activity.
TMP-INDUCED
487
STERILITY
Choline acetyltransferase activity of rabbit spermatozoa was measured (Fig. 6). A developmental pattern was observed. Lowest activity was measured in the caput, 8.0 pmol of acetylcholine (ACh) synthesized per 1 x lo5 spermatozoa per hour, with SPERMATOZOA
‘5
HOURS
POST
TREATMENT
FIG. 5. Acetylcholine synthesis in rat spermatozoa. Values represent the mean (*SE) picomoles of acetylcholine synthesized per lo5 rat spermatozoa per hour. Trimethylphosphate, 750 mg/kg, was administered and animals sacrificed at the specified time periods.
PROXIMAL CORPUS
DISTAL CORPUS
PROXIUAL CAUDA
DISTAL CAUOA
Fm. 6. Acetylcholine synthesis in rabbit spermatozoa. Values represent the mean (&SE) picomoles of acetylcholine synthesized per 10’ rabbit spermatozoa per hour. Trimethylphosphate, 750 mg/kg, was administered once and choline acetyltransferase measured 1 week following treatment. Trimethylphosphate, 200 and 325 mg/kg, was administered weekly for 12 weeks and choline acetyltransferase measured during that week of treatment. Post-treatment measurements of choline acetyltransferase were at 1 to 2 weeks following termination of treatment. Control animals received the same volume of vehicle.
increasing amounts measured in proximal and distal corpus and proximal cauda. Highest levels, six times greater than caput, were measured in the distal cauda, 35 pmol of ACh synthesized per 1 x lo5 spermatozoa per hour. A single TMP treatment reduced spermatozoa choline acetyltransferase activity from all segments of the epididymis, but significant differences were observed only in spermatozoa from proximal cauda and
488
HARBISON.
DWIVEDI
AND
EVANS
distal cauda. Continued weekly treatment with TMP maintained depression of choline acetyltransferase activity. A dose vs response depression of choline acetyltransferase activity was observed when a treatment of TMP of 325 mg/kg was compared to a dosage of 200 mg/kg. A dosage of 325 mg/kg in general produced a greater depression of enzyme activity. Continued TMP treatment at the lower dosages produced as great or greater reductions in choline acetyltransferase activity. TMP-induced reduction of this enzyme system was completely reversible. One to two weeks following termination of treatment, choline acetyltransferase activity returned to normal in spermatozoa from all segments of the epididymis (Fig. 6).
1L iBElT
AN
7. Acetylcholine synthesis in spermatozoa obtained from rats, rabbits, and humans. Values represent the mean (*SE) picomoles of acetylcholine synthesized per 10’ spermatozoa per hour. Rat and rabbit spermatozoa were obtained from the distal cauda; human spermatozoa were obtained fresh by ejaculation. FIG.
Spermatozoa choline acetyltransferase activity was measured in rat, rabbit, and man (Fig. 7). Human spermatozoa choline acetyltransferase activity was assayed to determine ifthis enzyme activity existed in human spermatozoa and to assessthe comparative activities of this sperm enzyme in the human and in laboratory animals. Rat spermatozoa contained the highest enzyme activity. Rabbit and human choline acetyltransferase activities were comparable, about one-half that of the rat. Spermatozoa of all three species contained nascent acetylcholine and were capable of synthesizing and releasing acetylcholine in vitro (Table 1). Human spermatozoa contained the highest amount of nascent ACh but not the highest choline acetyltransferase activity. However, human spermatozoa were capable of synthesizing and releasing, during a 30-min incubation, three to four times the quantity of ACh produced by rat and rabbit sperm.
TMP-INDUCED
489
STERILITY
TABLE 1 SPERMATOZOA ACETYLCHOLINE CONTENT IN SEVERAL SPECIES" Picomoles Species Rabbit Rat Man
Not incubated 18.5 t- 1.1 15.4 &- 1.2 30.6 + 1.9
of ACh/106
spermatozoa
15 minh
30 min
45 min
60 min
20.2 & 1.3 18.6 f 2.2 -
22.2 f 1.2 22.7 -t 2.1 79.3 + 7.1
25.0 + 0.7 32.7 + 2.1 -
31.1 + 0.3 41.1 It. 3.3 -
a Values are means k SE. Not incubated spermatozoa acetylcholine content represent nascent concentrations of acetylcholine. Incubation of spermatozoa with phospholine iodide (1 x low3 M) reflects synthesis and release of acetylcholine into the incubation medium. b Incubation time.
A concentration-dependent inhibition of spermatozoa motility was seen when TMP was added to a sperm suspension in vitro. TMP, 2.5 x lo-’ M, suppressed spermatozoa motility by about 21%. Also in vitro 1 x 10e5 M TMP, inhibited choline acetyltransferase purified from human placenta by about 32%. This was the maximal inhibition that could be obtained in vitro.
DISCUSSION
TMP induces reversible sterility in three species, mouse, rat, and rabbit. Sterility was dependent on dose and duration of treatment and was produced within 1 to 2 weeks following initiation of treatment and in all cases was reversible. Fertility returned to normal within 1 to 2 weeks after termination of treatment. Spermatogenesis was not affected by TMP treatment. TMP primarily affects epididymal spermatozoa, probably by an action on sperm motility. Rabbit spermatozoa were markedly depressed in motility after administration of TMP, and this depression was accompanied by a depression of choline acetyltransferase activity. Mammalian spermatozoa contain high acetylcholinesterase activity, which is concentrated in the flagella (Sekine, 1951; Nelson, 1964, 1966). Spermatozoa preferentially hydrolyze acetylcholine over butyryl- and benzoylcholine (Appelgate and Nelson, 1962; Nelson, 1964). Nelson has proposed a working hypothesis that the acetylcholinesterase may serve to regulate sperm motility by controlling intracellular concentrations of acetylcholine, The synthesis of acetylcholine is controlled by the enzyme choline acetyltransferase. The regulation of sperm motility by acetylcholine is therefore probably dependent on both the rate of acetylcholine synthesis by choline acetyltransferase and the rate of acetylcholine hydrolysis by acetylcholinesterase. These two enzymic actions will control the intracellular content of acetylcholine. Therefore, inhibition of choline acet)rltransferase activity should result in a drop in acetylcholine content and a resultant impairment of spermatozoa motility and ability to fertilize. From the present results, the mechanism of TMP-induced sterility appears to involve inhibition of the spermatozoa enzyme choline acetyltransferase. Inhibition of this enzyme compromises spermatozoa motility and ability to fertilize.
490
HARBISON.
DWIVEDI
AND
EVANS
TMP as well as other tri-alkyl phosphates may inhibit the spermatozoa enzyme choline acetyltransferase and thereby produce a functional sterility. The di-alkyl phosphates useful as organophosphorus pesticides may likewise produce a functional sterility because of their suspected inhibition of choline acetyltransferase. Such a possibility must be experimentally proven in each case. Choline acetyltransferase is present in the sperm of all three species studied, including man. This enzyme appears to be critical for the fertilizing capacity of spermatozoa. Temporary sterility of males exposed to occupational or environmental chemicals may possibly be explained by their effect on the spermatozoa enzyme choline acetyltransferase. The ability of the spermatozoa cholinergic system to regulate its fertilizing capability is a provocative finding. Development of tri-alkyl phosphates or di-alkyl phosphates as potent choline acetyltransferase inhibitors and potential male sterilants is suggested. REFERENCES APPLEGATE,
A.
AND
NELSON,
L. (1962). Acetylcholinesterase in mytilus spermatozoa. Biol.
Bull. 1X3,475-476.
J. H., RADIKE, A. AND MUNDY, B. W. (1942).J. Amer. Chem. Sot. 64,2977-2978. W. B. AND WITHERUP, S. (1946).Observation on effectsof trimethyl phosphate upon experimentalanimals.J. Pharmacol. Exp. Ther. 88, 338-342. GUMBMANN, M. R., GAGNE, W. E. AND WILLIAMS, S. W. (1968).Short-term toxicity studiesof rats fed triethyl phosphatein the diet. Toxicol. Appl. Pharmacol. 12, 360-371. HARBISON, R. D., OLUBADEWO, J., DWIVEDI, C. AND SASTRY, B. V. (1975).Proposedrole of the placentalcholinergicsystemin the regulationof fetal growth and development.In Basic and Therapeutic Aspects of Perinatal Pharmacology (P. L. Morselli, S. Garattini, and F. Sereni,Eds.). Raven Press,New York. HINKAMPS, J. B. AND WARREN, J. A. (1968).Ethyl Ignition Control Compound 4. A Gasoline Additive,for Control of Spark Plug Fouling, Surface Ignition and Rumble. Ethyl Corporation Technical Publication ICC 4. JACKSON, H. AND JONES, A. R. (1968).Antifertility action and metabolismof trimethylphosphate in rodents. Nature (London) 22, 591-592. JONES, A. R. (1970). The metabolismof tri-alkyl phosphates.Experientia 26, 492493. MCCAMAN, R. E., AND HUNT, J. M. (1965). Microdetermination of choline acetylasein nervoustissue.J. Neurochem. 12, 253-259. NELSON, L. (1964).Acetylcholinesterasein bull spermatozoa.J. Reprod. Fert. 7, 65-71. NELSON, L. (1966). Enzyme distribution in “naturally-decapitated” bull spermatozoa: Acetylcholinesterase, adenylpyrophosphataseand adenosinetriphosphatase.J. Cell. Physiol. 68, 113-l 16. SEKINE, T. (1951).Cholinesterase in pig spermatozoa.J. Biochem. (Tokyo) 38,171-179. TOY, A. D. R. (1949). Preparation of tetramethyl pyrophosphate.J. Amer. Chem. Sot. 71, 2268-2269. BILLMAN, DEICHMANN,
