The inhibition of rat and guinea pig cholinesterases by anionic hydrolysis products of methylphosphonic difluoride (difluoro)

561-566(1986)

TOXICOLOGYANDAPPLIEDPHARMACOLOGY~,

The Inhibition of Rat and Guinea Pig Cholinesterases Products of Methylphosphonic Difluoride

by Anionic Hydrolysis (Difluoro)

A. R. DAHL, C. H. HOBBS, AND T. C. MARSHALL Inhalation

Toxicology

Research Institute, Lovelace Biomedical and Environmental P.O. Box 5890. Albuquerque, New Mexico 87185

Received

September

20, 1985:

accepted

February

Research

Institute,

IO, 1986

The Inhibition of Rat and Guinea Pig Cholinesterases by Anionic Hydrolysis Products of Methylphosphonic Ditluoride (Difluoro). DAHL, A. R., HOBBS, C. H., AND MARSHALL, T. C. (1986). Toxicol. Appl. Pharmacol. 84, 56 l-566. Methylphosphonic difluoride (difluoro) and its hydrolysis products, methylphosphonfluoridate (MF) and fluoride, were examined for cholinesterase-inhibiting ability in rats and guinea pigs by both inhalation and intraperitoneal exposure routes. In vivo inhibition was compared to in vitro inhibition. In the whole animal, MF was the active chemical. but in vitro under special conditions, difluoro was more potent than MF and fluoride. Rat and guinea pig blood cholinesterases were equally sensitive to inhibition by MF, but only the guinea pig displayed cholinergic signs leading to death from MF toxicity. Data imply that MF is responsible for the cholinesterase inhibition resulting from exposure to DF vapor. MF may be the first example of a moderately strong acid shown to inhibit cholinesterase and cause death from cholinergic effects.

Difluoro is an intermediate in the preparation of potent cholinesterase inhibitors. It is rapidly hydrolysed in water to methylphosphonofluoridic acid (MF) and hydrogen fluoride (Beach and Sass, 1961). The MF, on the other hand, is only slowly hydrolyzed at physiological pH with a half-life of about 1000 hr (Bechtold and Dahl, 1986). Despite its short half-life in aqueous solution, difluoro has been reported to form complexes with aqueous solutions of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) causing inhibition of its enzymatic activity (Wins and Wilson, 1974). It has also been reported that prolonged (up to 52 weeks) inhalation of difluoro vapor at 1 mg/m3 decreased blood cholinesterase levels in dogs (McNamara et al., 1974). In the studies by Wins and Wilson, it was concluded that unhydrolyzed difluoro reacted directly with the cholinesterase to form an inhibited complex. This mechanism seemed unlikely in the in vivo situation reported by McNamara 561

et al., (1979), since difluoro is rapidly hydro-

lysed, but those results could have been explained as inhibition due to fluoride ion, a known weak inhibitor of cholinesterase (Usdin, 1970). During acute toxicity studies at this Institute with difluoro, MF, and fluoride ion, it was observed that MF apparently was a sufficiently active cholinesterase inhibitor to account for all the inhibition attributed to difluoro in the in vivo experiments. Further experiments were carried out to confirm initial observations and these experiments are the subject of this report. METHODS Chemicals. Laboratory quantities of difluoro were supplied by the U.S. Army Chemical Research and Development Center (Aberdeen, Md.). The difluoro was purified by distillation (BP 94-96°C at 640 mm Hg) from anhydrous sodium carbonate crystals. Purity was verified by phosphorus 3 1 nuclear magnetic resonance (NMR) spectroscopy and exceeded 98%.

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DAHL, HOBBS, AND MARSHALL

80

TREATMENT

FIG. 1. Inhibition of tat blood cholinesterase by difluoro in vitro. The assay was run as follows: whole rat blood, 150 ~1 in 50 ml phosphate buffer, was rapidly stirred at 37°C while 100 ~1 of a solution of difluoro in acetonitrile (1 mg DF/ml) was added. The final concentration of DF was 20 pM. The solution was assayed for chohnesterase after it was either immediately diluted and cooled (bar 3); allowed to stand 30 min (bar 4); treated immediately with PAM (final concentration 0.06 mM) (bar 5); or treated with PAM (same concentration) after 30 min (bar 6). Bar 1 is a control containing neither difluoro nor PAM. Bar 2 is control plus PAM (same concentration). N = 3. Error bars are standard errors. *Significantly different than bar 1 (p =Z0.05).

midity. The rooms were maintained on a 12-hr light, 12hr dark cycle, begining at 6:oO AM. Species-formulated food (Allied Mills, Chicago, Ill.) and water were available ad libitum. All animals’ cages were changed on a weekly basis. Esteruse assays.Cholinesterase assayson blood samples were carried out on whole blood using modifications of the method of Ellman (Ellman et a!., 1961). The modifications were made to automate assaysusing a Multistat III centrifugal analyzer (Instrumentation Laboratory, Lexington, Mass.). Cholinesterase assayson brain tissue were carried out on homogenates of whole brain according to the method of Ellman without modification using an Aminco DW-2A spectrophotometer (Travenol Labs). Data ana@sis. All data reduction and analyses were carried out on RS-I software on a VAX 1170 computer (Digital Equipment, Inc.). In vitro studies. Blood was drawn by heart puncture from pentobarbital anesthetized animals for in vitro studies. Difluoro was diluted in acetonitrile freshly distilled from phosphorus pentoxide so that a maximum of 100 ~1 of solution was added to 50 ml of diluted blood of 0.1 M postassium phosphate buffer (pH 7.4) (3 ~1 blood/ml of solution). MF as the sodium salt or tluoride as the sodium or potassium salt were added as solutions in distilled water at pH 7.4 (adjusted with HCl or NaOH). For experiments with PAM, the PAM was added as an aqueous solution in a total volume of 275 ~1.Concentrations of compounds used are detailed in the captions to Figs. 1 and 2. lOOr

60-

MF was prepared from ditluoro by hydrolysis with a cold solution of sodium bicarbonate. Fluoride ion was removed by precipitation as calcium fluoride, and the solute was analyzed by ion chromatography using a Dionex 202Oi ion chromatograph. Potassium fluoride dihydrate, sodium fluoride, bovine cholinesterase (erythrocyte AChE, EC 3.1. I .7), and pyridine-2aldoximine methiodide (PAM) were purchased from Sigma (St. Louis, MO.), as were all other chemicals used in the assay for cholinesterase. The PAM was used to test the reversibility of rat blood cholinesterase inhibited by difluoro in in vitro experiments. Animals. Specific-pathogen-free, male F344/N rats, 1520 weeks of age, reared at the Institute, were used in these studies. Male Hartley strain guinea pigs [Crl:(HA)BP] were purchased from Charles River (Wilmington, Mass.). Animals were purchased at ages 1l- 13 weeks and were maintained in quarantine for a minimum of 2 weeks. The animals were 13- 15 weeks old and weighed 350-500 g at the time of the experiments. Before use, animals of both species were housed two per filter-topped, polycarbonate cage, on sterilized hardwood-chip bedding. The animal rooms were maintained at 25°C with 40-60% relative hu-

0 k a i I

60-

F 0 5 Y

40-

% 20-

0

0.01

0.1 CONCENTRATION

1.0

10

100

OF MF hWlho,.r)

FIG. 2. Inhibition of purified bovine chohnesterase and rat and guinea pig blood cholinesterase in vitro by MF. Appropriate amounts of the sodium of potassium salt of MF were added to 50 ml of phosphate buffer (pH 7.4) containing 150 ~1 of whole rate or guinea pig blood or to a solution of 1.56 mU of purified bovine cholinesterase per 100 ~1 buffer. The Ellman assaywas carried out after - 10 min at 37°C. The r2 values for the assaywere >0.99 except for the two highest concentrations of MF with bovine enzyme (r* z 0.96). Standard errors were <4%.

CHOLINESTERASE

10

INHIBITION

100

1,000

HOURS AFTEREXPOSURE

FIG.3. Inhibition of blood cholinesterase in rats exposed I hr to difluoro vapor. Four or five rats were exposed to the indicated concentrations of difluoro vapor or clean air (sham exposure) for 1 hr. Blood samples were obtained from the ocular orbit at the indicated times after exposure and analysed for ChE activity by the Ellman method. Error bars are standard errors. In vivo studies. Animals were administered solutions of MF, fluoride, and equimolar concentrations of fluoride and MF (i.e., hydrolysed difluoro) as the sodium salts in water (pH 7.4) by intraperitoneal injection. For inhalation studies, rats were exposed to graded concentrations of difluoro vapor for 1 hr as described below. Dosages and air concentrations are detailed in Figs. 3 and 4 and the caption of Fig. 5. Following either inhalation or intraperitoneal administration, periodic IO+1 blood samples were drawn from the ocular orbital sinus and diluted in saline for cholinesterase assays.For brain cholinesterase assays,animals

170

BY DIFLUORO

563

were terminated by carbon dioxide asphyxiation at the desired posttreatment time and the whole brain was removed for homogenization in 15 ml of 0.1 Mpotassium phosphate buffer (pH 7.4) and diluted for the assayin the same buffer to a concentration of 5 mg/ml. DF generation and analysis of vapor. The difluoro was vaporized by use of a syringe placed in a J-tube made of Teflon and heated to 100°C (Miller et al., 1980). The animals were exposed for 1 hr to the difluoro vapor in a multitiered inhalation chamber (HC 2000, Hazleton Systems, Inc.). The animals were maintained in individual cages. The chamber had a volume of -2 m3. Total air flow through the chamber was 500 liters/mitt. The exposure atmosphere was sampled with two bubbler samples over the I-hr period. The bubbler samples were analyzed for fluoride ion and MF by ion chromatography. The chamber was also monitored for difluoro using a Miran 1A infrared spectrometer (Foxboro Instruments, Foxboro, Mass.) set at 10.6 pg.

RESULTS In vitro. Rat blood treated with difluoro in acetonitrile according to the method of Wins and Wilson ( 1974) was inhibited by about 40% when the final theoretical difluoro concentration was 20 PM (Fig. 1). The amount of inhibition was only marginally affected, if at all, by the amount of time allowed prior to cooling and diluting the solution to stop further reaction. Treatment with 0.06 M pyridine-2-aldoxime methiodide appeared to partly regenerate cholinesterase activity when added quickly (3-6 set) after the difluoro (DF) treat-

r

20 ’ 1

I 100

10 HOURS

AFTER

I 1,000

EXPOSURE

FIG.4. Inhibition of blood cholinestemse in rats administered dilluoro by inhalation, or MF or fluoride by intraperitoneal injection. Except for the intraperitoneal administrations (sodium salts in l-2 ml of water at pH 7.4) details were as described in the legend to Fig. I.

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DAHL,

HOBBS, AND MARSHALL

w 3 60 d E 459 LL 300 !i 0” 15!

0. 1

10

100

MINUTES

1.000

AFTER

4 10.000

INJECTION

FIG. 5. Inhibition of guinea pig blood cholinesterase after intraperitoneal administration of MF. Three guinea pigs were administered l-2 ml of MF as the sodium salt in water at pH 7.4. The dose was 30 mg MF/kg. Blood samples were drawn from the ocular orbit at the indicated time after administration. Cholinesterase assayswere by the Ellman method. Error bars are standard errors. N = 3.

ment. Longer delays (0.5 or 3 hr) between difluoro and PAM treatments appeared to decrease the amount of regenerated cholinesterase as illustrated for the 0.5-hr delay (Fig. 1). There was no substantive difference between results obtained after 0.5 and 3-hr delays. The apparent slight enhancement of cholinesterase activity by PAM alone as observed in this study was previously reported (Karlog and Peterson, 1963). Figure 2 shows the effect of MF alone on purified bovine erythrocyte AChE, and rat and guinea pig whole blood cholinesterase. There

was a clear dose/response relationship in the blood of both rodent species and the purified enzyme. The 50% inhibitions (150) of cholinesterase occurred between 0.5 and 5 mM for MF. For fluoride ion (data not shown) the 150 was between 10 and 50 mM. In vivo. Data from the in vivo experiments in rats are shown in Figs. 3 and 4. Figure 3 shows the dose/response relationship of rat blood cholinesterase after a I-hr exposure of rats to graded concentrations of difluoro vapor. Recovery to approximately normal levels occurred by about 100 hr. However, this may be misleading because sham treatment alone appeared to give a rebound effect for blood cholinesterase, possibly as a result of repetitive blood sampling. In Fig. 4. the data for the effect of an inhalation exposure of difluoro or intraperitoneal administrations of MF, sodium fluoride, and MF and sodium fluoride combined (hydrolyzed difluoro) are combined. MF and the hydrolyzed difluoro had similar effects whereas fluoride showed little effect, if any, at the concentration used, which was the equivalent to that derived from 10 mg/kg hydrolyzed difluoro. The results of intraperitoneal administration of MF in guinea pigs are shown in Fig. 5 and Table 1. Blood cholinesterase was not measured in guinea pigs exposed to difluoro because of deaths from reflex-mediated bronchiolar constrictions (Dahl et al., 1984). How-

TABLE 1 INHIBITION

Dose level b-a/k) Control 3 30 300

OF BLWD

AND BRAIN CHOLINESTERASE

Time to lacrimation (min) NO’ NOC 54 * 5 14 f 2

Time to death (min)’ Terminated Terminated Terminated 17*2

AFTER INTRAPERITONEAL

INJECTION

OF MP

Blood ChE (% control & SE)

Brain ChE (% control +- SE)

100 + 3.5 65 + 6.4 3 f 0.6

100 + 12.1 63 -+ 4.0 59 f 2.9 52 + 7.5

1 + 0.6

’ Guinea pigs administered MF sodium salt in saline (- 1 ml) by intraperitoneal injection (N = 3). ’ Surviving guinea pigs were exsanguinated after intraperitoneal injection of pentobarbital 60 min after treatment. At 300 mg/kg 3 of 3 animals died with typical cholinergic signs. ’ NO = not observed.

CHOLINESTERASE

INHIBITION

ever, guinea pigs given MF intraperitoneally exhibited obvious signs of cholinesterase inhibition (Table 1) but rats did not show obvious signs during any of the studies (Dahl et al., 1984). The time course for cholinesterase inhibition in the guinea pigs after intraperitoneal administration of MF is shown in Fig. 5. The onset of cholinergic effects (Table 1) and decreases in blood cholinesterase (Fig. 5) paralleled each other. DISCUSSION The data in Fig. 1 are consistent with the earlier reports (Wins and Wilson, 1974) showing that difluoro and similar compounds inhibit cholinesterase. Difluoro, under the rather special conditions used in the in vitro assay, was more potent than MF. This is illustrated by comparing Fig. 1, where 20 j&M DF inhibited 40% of the cholinesterase, with Fig. 2, where MF concentrations of about 1000 pM were needed to achieve the same response. The difference is probably due to binding of difluoro to serine on cholinesterase as suggested earlier (Wins and Wilson, 1974). Both rat and guinea pig blood cholinesterase and purified bovine cholinesterase exhibited 50% inhibition (150) at between 0.5 and 5 m&t MF (Fig. 2). The data in Fig. 3 show the dose/response relationship for blood cholinesterase after a lhr exposure of rats to graded concentrations of difluoro vapor. The blood concentrations of MF for the in vivo experiments in rats can be estimated for the inhalation exposures as follows. Assuming a minute volume of 0.180 liters/min (Mauderly et al., 1979), 100% deposition of inhaled difluoro vapor, and 100% absorption of the hydrolysis product, MF, into the blood (Dahl and Bechtold, 1985) and a total blood volume of 12 ml for a 200 g rat (Everett et al., 1956) then, if no elimination occurs until absorption is complete, the maximum concentration of MF after a I-hr exposure at 1000 pg DF/liter would be: 0.18 liter/ min X 60 min X 1000 pg DF/liter X 1 pmol DF/ 100 pg DF + 0.0 12 liter of blood = 9 mM.

BY

DIFLUORO

565

The MF blood concentration after difluoro exposure at concentrations of 600 and 400 pg/ liter would be proportionately less. Thus, the amount of inhibition of rat blood ChE after inhalation of difluoro (Fig. 3), roughly corresponds to that expected for MF in in vitro experiments (Fig. 2). Similar calculations using the data in Fig. 4 for intraperitoneal administration of MF give a maximum MF blood concentration after the 10 mg MF/kg dose of 1.7 mM. Again, the amount of inhibition observed (Fig. 4) corresponds with that predicted from in vitro experiments (Fig. 2). That MF is responsible for the observed cholinesterase inhibition in vivo seems well established by the data. That MF can cause cholinergic signs, including death, in guinea pigs is indicated by comparing the data in Table 1 with those in Fig. 5. The time of onset of cholinergic signs after intraperitoneal administration of 30 mg MF/kg (Table 1) corresponds with the time for maximum inhibition of blood cholinesterase (Fig. 5). The data in Table 1 also show inhibition of guinea pig brain cholinesterase which was maximum at the lowest tested dose. This effect was not observed after inhalation or interperitoneal exposure in rats and may, in part, account for the species differences in response to MF. However, the level of brain cholinesterase inhibition plateaued and doses of MF greater than 3 mg/kg did not produce proportionate decreases. In fact, blood cholinesterase levels appear to correspond better with the onset of cholinergic signs. To our knowledge, MF is the first example of an anion of a moderately strong acid (K, approximately 0.3) inhibiting cholinesterase and causing death due to cholinergic effects. Earlier reports ascribed difluoro cholinesterase inhibition to the direct reaction of difluoro with serine hydroxyl group on the cholinesterase molecule (Wins and Wilson, 1974). The possibility of inhibition by the hydrolysis product, MF, was not considered. However, the results reported here show that MF is the inhibiting species in vivo. In summary, we have found that inhalation

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of difluoro vapor at relatively high concentrations (2400 pg/liter) caused significant inhibition of rat blood cholinesterase, but caused neither measureable inhibition of rat brain cholinesterase nor obvious cholinergic signs. On the other hand, guinea pigs (which, due to susceptibility to bronchiolar constriction when exposed to high concentrations of difluoro were not exposed to difluoro by that route for the purpose of cholinesterase studies) exhibited sensitivity to cholinergic effects from the hydrolysis product of difluoro, MF. MF inhibited both brain and blood cholinesterase in guinea pigs. MF also inhibited purified bovine cholinesterase as well as rat and guinea pig blood cholinesterase in in vitro experiments. ACKNOWLEDGMENTS We thank Y. S. Cheng and B. V. Mokler for their assistance in developing exposure methods for difluoro, and J. Waide and T. Gugliotta for technical assistance. Research supported by the U.S. Army Chemical Research and Development Center, Aberdeen Proving Ground, under a memorandum of understanding with the Department of Energy under Contract DE-AC04-76EV0 1013 and in facilities fully accredited by the American Association of Laboratory Animal Care.

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BECHTOLD, W. E., AND DAHL, A. R. (1986). Kinetics and mechanism of hydrolysis of methylphosphonfluoridic acid (in press).

DAHL, A. R., MARSHALL, T. C., AND HOE=, C. H. ( 1984). The acute toxicity of an inhalable fluorophosphonate (difluoro) and chlorophosphonate (dichloro) in three species of rodents. Toxicologist 4,22. DAHL, A. R., AND BECHTOLD, W. E. (1985). Deposition and clearance of a water-reactive vapor, methylphosphonic difluoride (difluoro) inhaled by rats. Toxicol. Appl.

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81, 58-66.

ELLMAN, G. L., COURTNEY, K. D., ANDRE& V., AND FEATHERSTONE, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem.

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EVERETT, N. B.. SIMMONS, B., AND LASHER, E. P. (1956). Distribution of blood (Fe59) and plasma (113’) volumes of rats determined by liquid nitrogen freezing. Circ. Res. 4,4 19-424. KARLOG, 0.. AND PETERSEN,H. E. H. (1963). The influence of oximes on the acetylthiocholine hydrolysis rate. Biochem.

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ARCSL-TR-78023. Chemical Aberdeen Proving Ground, Md. MILLER, R. R.. LEGS, R. L.. POTTS, W. J. AND MCKENNA, M. J. (1980). Improved methodology for generating controlled test atmospheres. Amer. Ind. Hyg. Assoc. J. 41, 844-846.

USDIN, E. (I 970). Reactions of cholinesterases with substrates, inhibitors and reactivators. In International Enqclopedia of Pharmacology and Therapeutics (A. G. Karczmar, ed.), Sec. 13, Vol 1, pp. 188ff. Pergamon, New York. WINS, P., AND WILSON, I. B. (I 974). The inhibition of acetylcholinesterase by organophosphorus compounds containing a P-Cl bond. Biochem. Biophys. Acta 334, 137-145.