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Soman levels in kidney and urine following administration to rat, guinea pig, and marmoset
Life Sciences, Vol. 50, pp. 1057-1062 Printed in the USA
Pergamon Press
SOMANLEVElSINKIDNEYANDURINEFOLUXVINGADA4INWRA
TION To BAT,
GUINEA PIG, AND MARMOSET L.P.A. & Jong, H.P. Benschop, A. Due, C. Van Dijk, H.C. Trap, H.J. Van der Wiel*, and RPM Van Helden* Rins Maurits Laboratory TN0 and “Medical Biological Laboratory TNO, P.O.Box 45,2280 AA Bijswijk, The Netherlands (Received
in final form January 29, 1992)
Concentrations of C!&)P(*)-soman (1,2,2&meth lpropyl methylphosphonofluoridate) in urine ofanaes&ti& atropinixed and artt! ’ rcially ventilated rats, guinea pigs andmarmose*rwaedctcrmined1_4h~ivadministrationof1_6LDn,oftkagent and in the kidneys 1 h after iv adminiitration of 2-6 LDm %C(*)P(*)-soman. The concentrations ofthe toxic C(*)P(-)-isomers in both urine and kidneys of the rat were atleasttwocjadasofmagnindehigherthanthecarespondingkvelsindretwoothtr species. Belatively high urine concentrations were also found for C(*)P(&)-somanintoxicated (6 m) rats pretteated with the nontoxic soman analogue PDP (1,2,2trimethyl dimethylphosphinate), which considerably decreases the persistence of C(zl$‘o>soman in rats, or the carboxylesterase inhibitor CBDP [2-(o-ctesyl)-4H1:3:2-benxodioxaphoqhorin-2-oxi&]. The lethal effect brought about by intravesical administration of C(*)P(*)-soman in rata showed that the agent can easily be reabsorbed frwm the bladder. It is concluded, that this reabsorption does probably not explain the previously observed persistence and “late toxicity” of C(&)P(&)-somanin rats, although the amount of renally excreted C(*)P(-)-soman (ca. 1 96 of the administeted dose) should be sufficient for a toxicologically sign&ant effect. From investigations in rats challenged with 6 LDsg of the nerve agent C(*)P(*)-soman (1,2,2-trimethylpropyl methylphosphonofluoridate, Fig. 1). Wolthuis and coworkers (1,2) observed that the neuromuscular transmission of the diaphragm could initially be restored by treatment with the oxime HI-6 [ l-(4-amino-carbonylpyridinio)methoxymethyl-2-hydroxyiminomethylpyridinium dichloride]. The neuromuscular transmission, however, became completely teinhibited 3.5 h later and the animals died of a typical anticholiistemse poisoning (“late toxicity”).
FIG. 1
-
Chemical structme of C(*)P(*)-soman. The chiral C and P atoms ate denoted by an astetisk. These observations initiated a number of studies on the toxicokinetics of the steteoisomets of C(&)P(&)-soman in anaesthetixed, atropinized and artificially ventilated rats, guinea pigs and
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Soman Levels
1058
in Kidney
and Urine
vol.
50,
NO. 14,
1992
marmosets after intravenous administration of 2-6 LDso of the agent (3-5). From these studies it appeared that the concentrations in blood of the relatively nontoxic C(*)P(+)-isomers decrease within minutes to a very low level, whereas low, but toxicologically significant, levels of the highly toxic C(&)P(-)-isomers persist for periods of l-4 h. The elimination of C(&)P(&)-soman occurs almost completely by way of enzymatic and nonenzymatic hydrolysis and of covalent binding to carboxylesterases and other proteins, as a consequence of the high reactivity of the phosphorusfluorine bond. Earlier results further indicated a larger persistence of C(&)P(-)-soman in rats than in guinea pigs and marmosets at least at high doses. Moreover, pretreatment of the animals with the nontoxic structural analogue of soman PDP (1,2,2-trimethylpropyl dimethylphosphinate) considerably decreased the persistence of C(*)P(-)-soman in rats, but hardly affected this persistence in the two other species (4,6). Pretreatment with the compound also prevented reintoxication and death of rats that were poisoned with C(*)P(f)-soman at doses 2 6 LDso and treated with an oxime (7). In the present work we investigated whether differences in renal excretion of C(&)P(&)soman might explain these interspecies differences. Therefore, concentrations of intact C(&)P(rt)soman in kidney and urine of rats, guinea pigs, and marmosets, at l-4 h after C(f)P(f)-soman intoxication, were determined. In additional experiments with rats, it was investigated whether toxicologically significant reabsorption of C(&)P(*)- soman from the bladder can take place. Materials and Methods
Materials
C(&)P(*)-soman, 2.2~dimethylpropyl methylphosphonofluoridate (neopentyl sari@, 1.2,2trimethylpropyl dimethylphosphinate (PDP), and C(*)P(+)-[U-ZH] 1.2.2~trimethylpropyl methylphosphonofluoridate [C(&)P(+)-D13-soman] were prepared as described previously (8). 2-(o-Cresyl)-4H-l:3:2-benzodioxaphosphorin-2-oxide (CBDP) was prepared according to Eto et al. (9). 1,2,2-Trimethylpropyl t4Cmethylphosphonofluoridate [14C-C(lt)P(*)-soman] was a gift of the U.S. Army Medical Research Institute for Chemical Defense (Aberdeen proving Ground, MD) and had a specific activity of 2.18 TBq/mole. Ethyl acetate (zur Rtlckstandsanalyse) was procured form Merck (Darmstadt, Germany) and was distilled over a column packed with Dixon rings. All other chemicals were obtained commercially and were used without further purification.
Male albino Wistar (WAG/Rij) rats bred in the Medical Biological Laboratory TN0 under specified pathogen-free conditions, male albino outbred guinea pigs of the Dunkin-Hartley type (Charles River, Sultzfeld, West Germany), and male marmosets (Callithrix iacchu& obtained from the Primate Center TNO, Rijswijk, The Netherlands), ca. one year old, were used. The animals were allowed to eat and drink ad libitum, until they were deprived of food 16 h before the experiment. .
.
Detenn ammo f levels of soman stereoisomers in urine &P(f)-Soman and PDP were intravenously administered to anaesthetized, auwpinized, and artificially ventilated rats (n=6-8) as described earlier (4). CBDP (16 mg/kg), freshly dissolved in propylene glycol (16 mg/ml), was administered subcutaneously in the neck, 1 h before administration of the nerve agent. The penis was ligated after administration of C(f)P(f)-soman. Urine was collected with a syringe after exposum of the bladder at the end of the experiment. During the experiment the core temperature of the animals was kept at 37 ‘C by means of a thermistorcontrolled heating lamp. Experiments with guinea pigs and marmosets were performed in a similar manner except that guinea pigs (n=7) were anaesthetized with ketam+ hydrochloride (40 mg/kg, im) and acepmmazine (6.6 mg/kg, im) and C(*)P(*)-soman was admuustered via a jugular cannula. Marmosets (n=6) were anaesthetized with ketamine hydrochloride (100 mg/kg, im) and C(&)P(&)-soman was injected directly in the jugular vein. The doses of atropine sulphate administered to guinea pigs and marmosets were 17.4 and 10 mg/kg (ip), respectively. For work-up of urine samples, soman stereoisomers were stabilized by addition of acetate buffer containing aluminium sulphate, neopentyl sarin, and the internal standard C(&)P(+)-Dl3-
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Soman Levels in Kidney and Urine
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soman, before extraction over Sep-Pak Cl8 columns (Mtllipore, Waters Associates, Mass.) and desorption of the analytes with ethyl acetate, as described previously for blood samples (8). The soman stereotsomers were analyzed by gas chromatography (vide infra). Determination of t4C-C(*)P(&)-soman levels in kidney The animals were treated in a similar manner as described for the determination of soman stereoisomers levels in urine, except that the animals were intoxicated with *4C-C(f)P(f)-soman (in aqueous solution containing < 10 %I isopropanol) and the penis was not ligated. Guinea pigs (n=5) were anaestheuzed with 2.5 % halothane m Nfl/oxygen (62:38) followed by sodium hexobarbital (40 mg/kg, ip) and were intoxicated with t4C-C(*)P(f)-soman by mtravenous administration. One hour after administration of I4C-C(+)P(+-)-soman, as much blood as possible was collected via the carotid cannula, before the animal was sacrificed and the kidneys were removed. Homogenates (25 %, w/w) m salme were made with a glass/glass Potter-Elvehjem homogenizer. Intact soman isomers were extracted from approximately 3 g homogenate with toluene (6.75 ml) (5). Radoactivity in the toluene layer was measured m a Tri-carb 4430 liquid scinhllation spectrometer (United Technologies Packard) after addition of the Hionic-Fluor scintillation cocktail (Packard Instruments B.V., Gromngen, The Netherlands). The toluene extracts of some of the kidney homogenates were also analyzed by gas chromatography. Two groups of anaesthetized and atropimzed rats (vide supra) were used. The first group of rats (n=4) was inJected with PDP (6 5 mgikg, iv) 10 min prior to an injection with 1.4 LDsu C(+)P(+)-soman (116 pg.kg-l.ml-1) by way of a cannula inserted mto the empty bladder. The second group (n=4) was treated identically except that PDP treatment was substituted by saline injection. Before administration of C(f)P(f)-soman, the penis of each animal was ligated. Survival times of the animals were determined. eRinsin
an Anaestheuzed and atropinized rats (n=5) were intravenously injected with 6 LD5o C(&)P(*)soman and immdately treated with HI-6 (56 m@g, iv). The bladder of these animals was rinsed with salme and dramed contmuously by means of an mfusion apparatus connected with a cannula inserted into the bladder. The speed of mfuston was 6.9 or 12 ml/h. Control animals (n=lO) were treated similarly except for cannulation of the bladder and draining. The survival times of the animals were determined.
Gas chromatographic analyses were performed with a Carlo Erba HRGC 5300 equipped with an alkali flame tomzation detector, on-column injection system and a Chrompack Multiple Switchmg Intelligent Controller for two-dimensional gas chromatography, using helium as a carrier gas. Samples (5 3 jtl) were injected on the CPSil 8 CB fused silica pxecolumn (length, 10 m; i.d., 0.53 mm; film thickness, 6.4 pm; Chrompack), programmed from 87 to 122 ‘C at 5 “Urnin. The cut was collected from 5.5 to 8 min after injection. After a waitmg time of 2.5 min. during which the chromatographic system was cooled to 60 ‘C, the trapped cut was mJected on the fused silica Chirasil-Val analytical column (length, 25 m; i.d., 0.22 mm; film thickness, 0.13 pm; Chrompack). The analytical column separating all soman stereoisomers and the internal standard was programmed from 60 to 72 “C at 1 ‘CYmin. Samples of marmoset urine were analyzed on a Chirasil-Val column obtained from Applied Science as described by Benschop et al (4). On this column the C(+)P(+)and C(+)P(-)-isomers were not separated.
Concentrations of intact C(*)P(&)-soman in the kidneys of anaesthetized, atropinized and arttficially ventilated rats, guinea pigs and marmosets were ra&ometrically determined 1 h after iv administrauon of 2-6 LD5o of 14C-C(k)P(&)-soman. The concentrattons m rat kidney were much higher than those found in the kidneys of the two other species (Table I). In some of the samples of rat kidney, the relatively high soman concentrattons were confirmed with gas chromatographic analysis. Only the two C(k)P(-)-isomers were detected.
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1992
Concomitant with the kidney levels of *‘%I-C(*)P(*)-soman, the urine of rats contained relatively high levels of C(k)P(-)-soman at 4 h after administration of 6 LD50 of C(k)P(rt)-soman, whereas the urine levels in guinea pigs and marmosets at 3 and 2 h, respectively, after administration of an equitoxic dose of C(k)P(rt)-soman were at least three orders of magnitude lower (Table II). The latter urine levels were even at least two orders of magnitude lower than those for rats that were challenged with a similar absolute dose (Table II), i.e., for rats challenged with 1 LD5o C(k)P(k)soman and for rats challenged with 6 LD50 of the agent after pretreatment with the carboxylesterase inhibitor CBDP (10). This pretreatment reduces the number of covalent binding sites for soman isomers, thus increasing the susceptibility of the ammal to C(k)P(i)-soman intoxication. The presence of C(-)P(+)-soman but not of C(+)P(+)-soman in the rat urine indicates that the former isomer is not generated in the urine by epimerization at phosphorus of C(-)P(-)-soman. TABLE I Mean Concent~ons~ (* s.e.m.. n = 5) of Intact 14C-C(k)P(*>seman m Kxlney of Anawheuzed. Atmpmwed and ArtGiially Ventdated Rats. Gumea pigs and Marmows. 1 h after IVadmmwatmn of l‘k-C(+)P(?+soman Speclea
Meandaseb hU?kR)
Rat Gumea pig h4allllOsete
520 230 165 55 57
(6.3) (2 8) :z (5.7)
Soman concentration b2la twsue) 810 f lsoc 180 + 2Od 2.3 + 0.5 0.5 f 0.1 0.15 f 0.06
a Determinedradlomemcally. b The figures m parenthesesdenote the doses expressedas muluples of the LDsOvalues. c Mean value obtamed from gas chromatographlcanalysis of two samples was 950 rig/g hssue. d Mean value obtamed from gas chromatographlcanalysis of three samples was 180 rig/g tissue. eN=3
From toxicokinetic determinations, Benschop et al. (4) concluded that pretreatment with PDP decreases the persistence of C(f)(P(-)-soman in rat blood. The present data show that PDP pretreatment considerably decreases the amount of intact C(k)P(-)-soman excreted in urine, albeit that the concentrations in urine are still much higher than those found for guinea pigs and marmosets challenged with an equitoxic dose (Table II). After administration of C(f)P(*)-soman (1.4 LD50) into the bladder of PDP-pretreated or non-pretreated rats, all animals died withm 30 min. Apparently, reabsorption of the agent from the bladder can easily take place and is not affected by PDP pretreatment. Thorough rinsing of the bladder of rats with saline subsequent to intoxication with a dose equivalent to 6 LD5o of C!(f)P(f)-soman and treatment with III-6 resulted in a mean survival time of 3. Ml.9 h. Without drainage of the bladder, the mean survival time in similarly poisoned and treated rats was 7.6k0.6 h. The rate of drainage with saline did not affect the result. Rinsing and drainage of the bladder of naive animals continuously during a period of 6 h did not hamper them.
In an absolute sense, very low amounts of intact C(*)P(-)-soman were renally excreted after iv administration of C(lt)P(lt)-soman to rats, CBDP- and PDP-pretreated rats, guinea pigs and marmosets. At most ca. 1 8 of the amount administered to the animal, i.e., in the case of CBDPpretreated rats intoxicated with a dose equivalent to 6 LD5o. Probably, the main reason for excretion of these low amounts is the very rapid metabolism of the agent due to enzymatically and nonenzymancally catalyzed hydrolysis and to covalent binding to proteins, which also leads to rapid decrease of C(*)P(->-soman levels in blood after iv administration (5). This result is consistent with the scarcely available information on renal excretion of C(lt)P(k)-soman and related compounds. Lenz et al. (11) reported that 1 h after SCadmmistration of 14C-labelled C(lt)P(k)-soman more than
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1992
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TABLE II Concentrauonsa of Soman Sterecnsomers m Urme Collected l-4 h afterIV Adnumstrationof C(i)(P(i)-soman to Anaesthetrzed, Atropmwxl and ArWkally VentWed Rats, Guinea pigs and Marmosets.
Concenmonsa Species
m Blood at 1 min afte.rIntoxlcanonObtamed from Previous Studies are mcluded.
Doseb Time after admmrShaUon (h)
0
Amount of urme in bl&W
(ml)
495
(6)
4
83
(1)
1
38
(6)
3
0 5aO 06 (8)
Rat/pDP8
495
(6)
4
0 5ofO 06 (7)
Gumeapig
165
(6)
3
1 If 0 3 (7)
Matm0se.t
60
(6)
2
Rat/CBDPf
038kOO7 (6) 03f
0 1 (6)
17* 06(6)
(@ml)
(@ml) C(-)P(+)
Rat
Concenmone of soman stereo~mers m blood lmmafter admmtstraaon
ConcentraUonc*d of soman stereoisomersin unne
17f6 5fl 1 lf0 2 o 5fo 4 _h 006foo3
C(+)P(-)
C(-)P(-)
(6)
134f19
136f20
42k7 44f7
(6)
6f4
14f3
(8)
14f2
14f2
19f8
(7)
128f17
121f13
0 04fO 01 0 08f0.03 (7)
49f6
59f6
OllfO05
35f4
31f2
C(+)P(-) 286f71 50f8 40f6 36fll
C(-)P(-) 212f68
013M05
(6)
aDetennmed v&h gas chromatography b The figures m parenthesesdenote the doses expressed as muluples of the LDsOvalues. c Means k s e.m.; n ISgiven m parentheses. d C(+)P(+)-somanwas not detected or, m the case of the marmoset urine. the concentrationsof the [C(+)P(+)-and C(+)P(-)]-isomerswere measured, smce these two isomers were not resolved by the gas chromatographlcanalysis used e Data from previous studies, see refs 4-7. f Rats were pretreatedwith CBDP (16 mg/kg, SC)1 h pnor to C(zk)P(k)-somanadmmlstra~on. g Rats were pretreatedwith PDP (6.4 mg/kg, IV)10 mm pnor to C(k)P(k)-soman admmlstration. h Not detected.
99 % of the renally excreted labelled compounds was hydrolyzed, whereas no intact compound was found by Heilbronn et al. (12) in the urine of rats after iv administration of tabun (ethyl N,Ndimethylphosphoramidocyanidate). Even PDP, which is chemically much more stable than C(rt)P(&)-soman, is renally excreted in rats for less than 1 9%as the intact compound (13). Only after intoxication with the organophosphate DPP (diisopropyl phosphorofluoridate), 5-10 % of the labelled compounds that were renally excreted during the first hour after intoxication of guinea pigs (14) and cats (15) could be detected as unaltered agent. Although intact C(lt>P(-)-soman is renally excreted m the three species to a slight extent only, the rat urine levels, and concomitantly the rat kidney levels, are at least two orders of magnitude higher than the corresponding levels in the two other species after administration of both equitoxic and similar absolute doses, whereas the blood levels found in the three species shortly (1 min) after intoxication with an eqmtoxic dose (6 LD5o) differ less than a factor of five (Table II; refs 4,5). The levels of the toxic C(&)P(-)-isomers in rat urine determined 1 or 4 h after intoxication with 1 or 6 LDso, respectively, even surpass the blood levels found 1 min after administration of the corresponding doses (Table II). This may indicate that most of the C(&)P(-)-soman is excreted within the first few minutes after intoxication. The presence of C(-)P(+)-soman in the urine, while this isomer disappears from the blood within a few minutes due to rapid hydrolysis by phosphorylphosphatases, provides additional support to this suggestion. It also indicates that phosphorylphosphatase activity is probably absent in urine and in the bladder. The presence of relatively high amounts of C(f)P(-)-soman in rat kidney 1 h after intoxication m spite of high binding capacity is somewhat puzzling. Apparently, the soman isomers are protected m the rat kidney against metabolic elimination. Tlus protection may be achieved when
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Soman Levels in Kidney and Urine
Vol. 50, NO. 14, 1992
the C(&)P(-)-soman in the kidney is, in fact, present m urine that has not yet been transported to the bladder. It is not clear, however, why this rapid excretion should occur in rats, but not in guinea pigs or marmosets. It has been observed (1) that C(&)P(*)-soman-intoxicated (2 6 LD50) rats died several hours after initial recovery due to oxtme treatment (“late toxicity”). The relatively high persistence of C(+)P(-)-soman in rats was confirmed by our previous toxicokinetic studies (5), which also indicated a larger persistence of the C(&)P(-)-isomers m rats than in guinea pigs and marmosets. Pretreatment with PDP considerably decreases the persistence of the isomers in rats, but has hardly any effect m this respect on the two other species (4,6). Previously, we have reasoned with regard to the persistence of C(+)P(-)-soman after a lethal dose, that areas under the curve down to approximately 1.8 ng.ml-l.mm may still lead to reinhibinon of a crmcal amount of the target enzyme acetylcholmesterase which has been reactivated, for example, by oximes or spontaneously (4). In this respect, the amount of renally excreted C(f)P(-)-soman m rats should be sufficient to be of toxicological stgnificance if the agent could be reabsorbed from the bladder. The lethal effect brought about by mtravestcal admmutratton of C(f)P(f)-soman showed that this process can Indeed take place. Therefore, at first glance it might be suggested that the higher persistence in rats than in the two other species can be understood on the basts of the relatively high C(+)P(-)-soman levels found in urme and kidneys However, survival times of mtoxicated rats, whtch were immediately treated with the therapeuttc agent HI-6, stgmficantly decreased when accumulation of this C(&)P(-)-soman depot is prevented by rinsing and drainage of the bladder. Conversely, in rats intoxicated with 1 LD50 C(f)P(+)-soman and m PDP-pretreated rats intoxicated with 6 LD5o C(+)P(f)-soman and subsequently treated with HI-6, much higher urine levels of C(+)P(-)-soman were found than in similarly treated guinea pigs and marmosets, whereas these rats did not show late toxicity. Therefore, whatever the importance of a certam amount of C(+)P(-)-soman reabsorbed from the bladder for its persistence in rats, it is concluded that it does not explain late toxicity found for the agent in this species. References
7. 8. 9. 10. 11. 12. ::15:
O.L. WOLTHUIS, F. BERENDS and E. MEETER, Fundam Appl. Toxicol. 1. 183-192 (1981). H.P.M. VAN HELDEN and O.L. WOLTHUIS, Eur. J. Pharmacol. 89 271-274 (1983). H P BENSCHOP, F. BERENDS and L.P A. DE JONG, Fundam. Appl. Toxicol. 1 177182 (1981). H.P. BENSCHOP, E.C. BIJLEVELD, L P A. DE JONG, H.J. VAN DER WIEL and H.P.M. VAN HELDEN, Toxicol. Appl. Pharmacol B 490-500 (1987). H.P. BENSCHOP and L.P.A. DE JONG, Neurosci. Biobehav. Rev. 15.73-77 (1991). H.P.M. VAN HELDEN, H.J. VAN DER WIEL and 0 L. WOLTHUIS, J. Pharm. Pharmacol. a 35-41 (1988). H.P.M. VAN HELDEN, H.P. BENSCHOP and O.L. WOLTHUIS, J. Pharm. Pharmacol. 38 19-23 (1986). H.P. BENSCHOP, E.C. BIJLEVELD, M.F. OTTO, C.E A.M. DEGENHARDT, H.P.M. VAN HELDEN and L.P.A. DE JONG, Anal. Bmchem. 151242-253 (1985). M. ETO, J.E. CASIDA and T. ETO, B&hem. Pharmacol. e 337-352 (1962). D.M. MAXWELL, K.M. BRECHT and B.L. O’NEILL, Toxtcol. Lett. 29 35-42 (1987). D.E. LENZ, D.M. MAXWELL, R. PRATHER and L BALL, The Pharmacologist 25 111, (1983). E. HEILBRONN, I.-E. APPELGREN and A. SUNDWALL, Biochem. Pharmacol. ti 11891195 (1964). H.P. BENSCHOP and H.C. WESSELMAN, Arch. Toxicol. a 238-243 (1989). D. HANSEN, E. SCHAUM and 0. WASSERMAN, Arch.Toxicol. a 73-81 (1968). D. HANSEN, E. SCHAUM and 0. WASSERMAN, Biochem. Pharmacol. 11 1159-1162 (1968).
Pergamon Press
SOMANLEVElSINKIDNEYANDURINEFOLUXVINGADA4INWRA
TION To BAT,
GUINEA PIG, AND MARMOSET L.P.A. & Jong, H.P. Benschop, A. Due, C. Van Dijk, H.C. Trap, H.J. Van der Wiel*, and RPM Van Helden* Rins Maurits Laboratory TN0 and “Medical Biological Laboratory TNO, P.O.Box 45,2280 AA Bijswijk, The Netherlands (Received
in final form January 29, 1992)
Concentrations of C!&)P(*)-soman (1,2,2&meth lpropyl methylphosphonofluoridate) in urine ofanaes&ti& atropinixed and artt! ’ rcially ventilated rats, guinea pigs andmarmose*rwaedctcrmined1_4h~ivadministrationof1_6LDn,oftkagent and in the kidneys 1 h after iv adminiitration of 2-6 LDm %C(*)P(*)-soman. The concentrations ofthe toxic C(*)P(-)-isomers in both urine and kidneys of the rat were atleasttwocjadasofmagnindehigherthanthecarespondingkvelsindretwoothtr species. Belatively high urine concentrations were also found for C(*)P(&)-somanintoxicated (6 m) rats pretteated with the nontoxic soman analogue PDP (1,2,2trimethyl dimethylphosphinate), which considerably decreases the persistence of C(zl$‘o>soman in rats, or the carboxylesterase inhibitor CBDP [2-(o-ctesyl)-4H1:3:2-benxodioxaphoqhorin-2-oxi&]. The lethal effect brought about by intravesical administration of C(*)P(*)-soman in rata showed that the agent can easily be reabsorbed frwm the bladder. It is concluded, that this reabsorption does probably not explain the previously observed persistence and “late toxicity” of C(&)P(&)-somanin rats, although the amount of renally excreted C(*)P(-)-soman (ca. 1 96 of the administeted dose) should be sufficient for a toxicologically sign&ant effect. From investigations in rats challenged with 6 LDsg of the nerve agent C(*)P(*)-soman (1,2,2-trimethylpropyl methylphosphonofluoridate, Fig. 1). Wolthuis and coworkers (1,2) observed that the neuromuscular transmission of the diaphragm could initially be restored by treatment with the oxime HI-6 [ l-(4-amino-carbonylpyridinio)methoxymethyl-2-hydroxyiminomethylpyridinium dichloride]. The neuromuscular transmission, however, became completely teinhibited 3.5 h later and the animals died of a typical anticholiistemse poisoning (“late toxicity”).
FIG. 1
-
Chemical structme of C(*)P(*)-soman. The chiral C and P atoms ate denoted by an astetisk. These observations initiated a number of studies on the toxicokinetics of the steteoisomets of C(&)P(&)-soman in anaesthetixed, atropinized and artificially ventilated rats, guinea pigs and
0024~3205/92
$5.00
Copyright 0 1992 Perganwn Preen plc
+
.oo All rights reserved.
Soman Levels
1058
in Kidney
and Urine
vol.
50,
NO. 14,
1992
marmosets after intravenous administration of 2-6 LDso of the agent (3-5). From these studies it appeared that the concentrations in blood of the relatively nontoxic C(*)P(+)-isomers decrease within minutes to a very low level, whereas low, but toxicologically significant, levels of the highly toxic C(&)P(-)-isomers persist for periods of l-4 h. The elimination of C(&)P(&)-soman occurs almost completely by way of enzymatic and nonenzymatic hydrolysis and of covalent binding to carboxylesterases and other proteins, as a consequence of the high reactivity of the phosphorusfluorine bond. Earlier results further indicated a larger persistence of C(&)P(-)-soman in rats than in guinea pigs and marmosets at least at high doses. Moreover, pretreatment of the animals with the nontoxic structural analogue of soman PDP (1,2,2-trimethylpropyl dimethylphosphinate) considerably decreased the persistence of C(*)P(-)-soman in rats, but hardly affected this persistence in the two other species (4,6). Pretreatment with the compound also prevented reintoxication and death of rats that were poisoned with C(*)P(f)-soman at doses 2 6 LDso and treated with an oxime (7). In the present work we investigated whether differences in renal excretion of C(&)P(&)soman might explain these interspecies differences. Therefore, concentrations of intact C(&)P(rt)soman in kidney and urine of rats, guinea pigs, and marmosets, at l-4 h after C(f)P(f)-soman intoxication, were determined. In additional experiments with rats, it was investigated whether toxicologically significant reabsorption of C(&)P(*)- soman from the bladder can take place. Materials and Methods
Materials
C(&)P(*)-soman, 2.2~dimethylpropyl methylphosphonofluoridate (neopentyl sari@, 1.2,2trimethylpropyl dimethylphosphinate (PDP), and C(*)P(+)-[U-ZH] 1.2.2~trimethylpropyl methylphosphonofluoridate [C(&)P(+)-D13-soman] were prepared as described previously (8). 2-(o-Cresyl)-4H-l:3:2-benzodioxaphosphorin-2-oxide (CBDP) was prepared according to Eto et al. (9). 1,2,2-Trimethylpropyl t4Cmethylphosphonofluoridate [14C-C(lt)P(*)-soman] was a gift of the U.S. Army Medical Research Institute for Chemical Defense (Aberdeen proving Ground, MD) and had a specific activity of 2.18 TBq/mole. Ethyl acetate (zur Rtlckstandsanalyse) was procured form Merck (Darmstadt, Germany) and was distilled over a column packed with Dixon rings. All other chemicals were obtained commercially and were used without further purification.
Male albino Wistar (WAG/Rij) rats bred in the Medical Biological Laboratory TN0 under specified pathogen-free conditions, male albino outbred guinea pigs of the Dunkin-Hartley type (Charles River, Sultzfeld, West Germany), and male marmosets (Callithrix iacchu& obtained from the Primate Center TNO, Rijswijk, The Netherlands), ca. one year old, were used. The animals were allowed to eat and drink ad libitum, until they were deprived of food 16 h before the experiment. .
.
Detenn ammo f levels of soman stereoisomers in urine &P(f)-Soman and PDP were intravenously administered to anaesthetized, auwpinized, and artificially ventilated rats (n=6-8) as described earlier (4). CBDP (16 mg/kg), freshly dissolved in propylene glycol (16 mg/ml), was administered subcutaneously in the neck, 1 h before administration of the nerve agent. The penis was ligated after administration of C(f)P(f)-soman. Urine was collected with a syringe after exposum of the bladder at the end of the experiment. During the experiment the core temperature of the animals was kept at 37 ‘C by means of a thermistorcontrolled heating lamp. Experiments with guinea pigs and marmosets were performed in a similar manner except that guinea pigs (n=7) were anaesthetized with ketam+ hydrochloride (40 mg/kg, im) and acepmmazine (6.6 mg/kg, im) and C(*)P(*)-soman was admuustered via a jugular cannula. Marmosets (n=6) were anaesthetized with ketamine hydrochloride (100 mg/kg, im) and C(&)P(&)-soman was injected directly in the jugular vein. The doses of atropine sulphate administered to guinea pigs and marmosets were 17.4 and 10 mg/kg (ip), respectively. For work-up of urine samples, soman stereoisomers were stabilized by addition of acetate buffer containing aluminium sulphate, neopentyl sarin, and the internal standard C(&)P(+)-Dl3-
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Soman Levels in Kidney and Urine
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soman, before extraction over Sep-Pak Cl8 columns (Mtllipore, Waters Associates, Mass.) and desorption of the analytes with ethyl acetate, as described previously for blood samples (8). The soman stereotsomers were analyzed by gas chromatography (vide infra). Determination of t4C-C(*)P(&)-soman levels in kidney The animals were treated in a similar manner as described for the determination of soman stereoisomers levels in urine, except that the animals were intoxicated with *4C-C(f)P(f)-soman (in aqueous solution containing < 10 %I isopropanol) and the penis was not ligated. Guinea pigs (n=5) were anaestheuzed with 2.5 % halothane m Nfl/oxygen (62:38) followed by sodium hexobarbital (40 mg/kg, ip) and were intoxicated with t4C-C(*)P(f)-soman by mtravenous administration. One hour after administration of I4C-C(+)P(+-)-soman, as much blood as possible was collected via the carotid cannula, before the animal was sacrificed and the kidneys were removed. Homogenates (25 %, w/w) m salme were made with a glass/glass Potter-Elvehjem homogenizer. Intact soman isomers were extracted from approximately 3 g homogenate with toluene (6.75 ml) (5). Radoactivity in the toluene layer was measured m a Tri-carb 4430 liquid scinhllation spectrometer (United Technologies Packard) after addition of the Hionic-Fluor scintillation cocktail (Packard Instruments B.V., Gromngen, The Netherlands). The toluene extracts of some of the kidney homogenates were also analyzed by gas chromatography. Two groups of anaesthetized and atropimzed rats (vide supra) were used. The first group of rats (n=4) was inJected with PDP (6 5 mgikg, iv) 10 min prior to an injection with 1.4 LDsu C(+)P(+)-soman (116 pg.kg-l.ml-1) by way of a cannula inserted mto the empty bladder. The second group (n=4) was treated identically except that PDP treatment was substituted by saline injection. Before administration of C(f)P(f)-soman, the penis of each animal was ligated. Survival times of the animals were determined. eRinsin
an Anaestheuzed and atropinized rats (n=5) were intravenously injected with 6 LD5o C(&)P(*)soman and immdately treated with HI-6 (56 m@g, iv). The bladder of these animals was rinsed with salme and dramed contmuously by means of an mfusion apparatus connected with a cannula inserted into the bladder. The speed of mfuston was 6.9 or 12 ml/h. Control animals (n=lO) were treated similarly except for cannulation of the bladder and draining. The survival times of the animals were determined.
Gas chromatographic analyses were performed with a Carlo Erba HRGC 5300 equipped with an alkali flame tomzation detector, on-column injection system and a Chrompack Multiple Switchmg Intelligent Controller for two-dimensional gas chromatography, using helium as a carrier gas. Samples (5 3 jtl) were injected on the CPSil 8 CB fused silica pxecolumn (length, 10 m; i.d., 0.53 mm; film thickness, 6.4 pm; Chrompack), programmed from 87 to 122 ‘C at 5 “Urnin. The cut was collected from 5.5 to 8 min after injection. After a waitmg time of 2.5 min. during which the chromatographic system was cooled to 60 ‘C, the trapped cut was mJected on the fused silica Chirasil-Val analytical column (length, 25 m; i.d., 0.22 mm; film thickness, 0.13 pm; Chrompack). The analytical column separating all soman stereoisomers and the internal standard was programmed from 60 to 72 “C at 1 ‘CYmin. Samples of marmoset urine were analyzed on a Chirasil-Val column obtained from Applied Science as described by Benschop et al (4). On this column the C(+)P(+)and C(+)P(-)-isomers were not separated.
Concentrations of intact C(*)P(&)-soman in the kidneys of anaesthetized, atropinized and arttficially ventilated rats, guinea pigs and marmosets were ra&ometrically determined 1 h after iv administrauon of 2-6 LD5o of 14C-C(k)P(&)-soman. The concentrattons m rat kidney were much higher than those found in the kidneys of the two other species (Table I). In some of the samples of rat kidney, the relatively high soman concentrattons were confirmed with gas chromatographic analysis. Only the two C(k)P(-)-isomers were detected.
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1992
Concomitant with the kidney levels of *‘%I-C(*)P(*)-soman, the urine of rats contained relatively high levels of C(k)P(-)-soman at 4 h after administration of 6 LD50 of C(k)P(rt)-soman, whereas the urine levels in guinea pigs and marmosets at 3 and 2 h, respectively, after administration of an equitoxic dose of C(k)P(rt)-soman were at least three orders of magnitude lower (Table II). The latter urine levels were even at least two orders of magnitude lower than those for rats that were challenged with a similar absolute dose (Table II), i.e., for rats challenged with 1 LD5o C(k)P(k)soman and for rats challenged with 6 LD50 of the agent after pretreatment with the carboxylesterase inhibitor CBDP (10). This pretreatment reduces the number of covalent binding sites for soman isomers, thus increasing the susceptibility of the ammal to C(k)P(i)-soman intoxication. The presence of C(-)P(+)-soman but not of C(+)P(+)-soman in the rat urine indicates that the former isomer is not generated in the urine by epimerization at phosphorus of C(-)P(-)-soman. TABLE I Mean Concent~ons~ (* s.e.m.. n = 5) of Intact 14C-C(k)P(*>seman m Kxlney of Anawheuzed. Atmpmwed and ArtGiially Ventdated Rats. Gumea pigs and Marmows. 1 h after IVadmmwatmn of l‘k-C(+)P(?+soman Speclea
Meandaseb hU?kR)
Rat Gumea pig h4allllOsete
520 230 165 55 57
(6.3) (2 8) :z (5.7)
Soman concentration b2la twsue) 810 f lsoc 180 + 2Od 2.3 + 0.5 0.5 f 0.1 0.15 f 0.06
a Determinedradlomemcally. b The figures m parenthesesdenote the doses expressedas muluples of the LDsOvalues. c Mean value obtamed from gas chromatographlcanalysis of two samples was 950 rig/g hssue. d Mean value obtamed from gas chromatographlcanalysis of three samples was 180 rig/g tissue. eN=3
From toxicokinetic determinations, Benschop et al. (4) concluded that pretreatment with PDP decreases the persistence of C(f)(P(-)-soman in rat blood. The present data show that PDP pretreatment considerably decreases the amount of intact C(k)P(-)-soman excreted in urine, albeit that the concentrations in urine are still much higher than those found for guinea pigs and marmosets challenged with an equitoxic dose (Table II). After administration of C(f)P(*)-soman (1.4 LD50) into the bladder of PDP-pretreated or non-pretreated rats, all animals died withm 30 min. Apparently, reabsorption of the agent from the bladder can easily take place and is not affected by PDP pretreatment. Thorough rinsing of the bladder of rats with saline subsequent to intoxication with a dose equivalent to 6 LD5o of C!(f)P(f)-soman and treatment with III-6 resulted in a mean survival time of 3. Ml.9 h. Without drainage of the bladder, the mean survival time in similarly poisoned and treated rats was 7.6k0.6 h. The rate of drainage with saline did not affect the result. Rinsing and drainage of the bladder of naive animals continuously during a period of 6 h did not hamper them.
In an absolute sense, very low amounts of intact C(*)P(-)-soman were renally excreted after iv administration of C(lt)P(lt)-soman to rats, CBDP- and PDP-pretreated rats, guinea pigs and marmosets. At most ca. 1 8 of the amount administered to the animal, i.e., in the case of CBDPpretreated rats intoxicated with a dose equivalent to 6 LD5o. Probably, the main reason for excretion of these low amounts is the very rapid metabolism of the agent due to enzymatically and nonenzymancally catalyzed hydrolysis and to covalent binding to proteins, which also leads to rapid decrease of C(*)P(->-soman levels in blood after iv administration (5). This result is consistent with the scarcely available information on renal excretion of C(lt)P(k)-soman and related compounds. Lenz et al. (11) reported that 1 h after SCadmmistration of 14C-labelled C(lt)P(k)-soman more than
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TABLE II Concentrauonsa of Soman Sterecnsomers m Urme Collected l-4 h afterIV Adnumstrationof C(i)(P(i)-soman to Anaesthetrzed, Atropmwxl and ArWkally VentWed Rats, Guinea pigs and Marmosets.
Concenmonsa Species
m Blood at 1 min afte.rIntoxlcanonObtamed from Previous Studies are mcluded.
Doseb Time after admmrShaUon (h)
0
Amount of urme in bl&W
(ml)
495
(6)
4
83
(1)
1
38
(6)
3
0 5aO 06 (8)
Rat/pDP8
495
(6)
4
0 5ofO 06 (7)
Gumeapig
165
(6)
3
1 If 0 3 (7)
Matm0se.t
60
(6)
2
Rat/CBDPf
038kOO7 (6) 03f
0 1 (6)
17* 06(6)
(@ml)
(@ml) C(-)P(+)
Rat
Concenmone of soman stereo~mers m blood lmmafter admmtstraaon
ConcentraUonc*d of soman stereoisomersin unne
17f6 5fl 1 lf0 2 o 5fo 4 _h 006foo3
C(+)P(-)
C(-)P(-)
(6)
134f19
136f20
42k7 44f7
(6)
6f4
14f3
(8)
14f2
14f2
19f8
(7)
128f17
121f13
0 04fO 01 0 08f0.03 (7)
49f6
59f6
OllfO05
35f4
31f2
C(+)P(-) 286f71 50f8 40f6 36fll
C(-)P(-) 212f68
013M05
(6)
aDetennmed v&h gas chromatography b The figures m parenthesesdenote the doses expressed as muluples of the LDsOvalues. c Means k s e.m.; n ISgiven m parentheses. d C(+)P(+)-somanwas not detected or, m the case of the marmoset urine. the concentrationsof the [C(+)P(+)-and C(+)P(-)]-isomerswere measured, smce these two isomers were not resolved by the gas chromatographlcanalysis used e Data from previous studies, see refs 4-7. f Rats were pretreatedwith CBDP (16 mg/kg, SC)1 h pnor to C(zk)P(k)-somanadmmlstra~on. g Rats were pretreatedwith PDP (6.4 mg/kg, IV)10 mm pnor to C(k)P(k)-soman admmlstration. h Not detected.
99 % of the renally excreted labelled compounds was hydrolyzed, whereas no intact compound was found by Heilbronn et al. (12) in the urine of rats after iv administration of tabun (ethyl N,Ndimethylphosphoramidocyanidate). Even PDP, which is chemically much more stable than C(rt)P(&)-soman, is renally excreted in rats for less than 1 9%as the intact compound (13). Only after intoxication with the organophosphate DPP (diisopropyl phosphorofluoridate), 5-10 % of the labelled compounds that were renally excreted during the first hour after intoxication of guinea pigs (14) and cats (15) could be detected as unaltered agent. Although intact C(lt>P(-)-soman is renally excreted m the three species to a slight extent only, the rat urine levels, and concomitantly the rat kidney levels, are at least two orders of magnitude higher than the corresponding levels in the two other species after administration of both equitoxic and similar absolute doses, whereas the blood levels found in the three species shortly (1 min) after intoxication with an eqmtoxic dose (6 LD5o) differ less than a factor of five (Table II; refs 4,5). The levels of the toxic C(&)P(-)-isomers in rat urine determined 1 or 4 h after intoxication with 1 or 6 LDso, respectively, even surpass the blood levels found 1 min after administration of the corresponding doses (Table II). This may indicate that most of the C(&)P(-)-soman is excreted within the first few minutes after intoxication. The presence of C(-)P(+)-soman in the urine, while this isomer disappears from the blood within a few minutes due to rapid hydrolysis by phosphorylphosphatases, provides additional support to this suggestion. It also indicates that phosphorylphosphatase activity is probably absent in urine and in the bladder. The presence of relatively high amounts of C(f)P(-)-soman in rat kidney 1 h after intoxication m spite of high binding capacity is somewhat puzzling. Apparently, the soman isomers are protected m the rat kidney against metabolic elimination. Tlus protection may be achieved when
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the C(&)P(-)-soman in the kidney is, in fact, present m urine that has not yet been transported to the bladder. It is not clear, however, why this rapid excretion should occur in rats, but not in guinea pigs or marmosets. It has been observed (1) that C(&)P(*)-soman-intoxicated (2 6 LD50) rats died several hours after initial recovery due to oxtme treatment (“late toxicity”). The relatively high persistence of C(+)P(-)-soman in rats was confirmed by our previous toxicokinetic studies (5), which also indicated a larger persistence of the C(&)P(-)-isomers m rats than in guinea pigs and marmosets. Pretreatment with PDP considerably decreases the persistence of the isomers in rats, but has hardly any effect m this respect on the two other species (4,6). Previously, we have reasoned with regard to the persistence of C(+)P(-)-soman after a lethal dose, that areas under the curve down to approximately 1.8 ng.ml-l.mm may still lead to reinhibinon of a crmcal amount of the target enzyme acetylcholmesterase which has been reactivated, for example, by oximes or spontaneously (4). In this respect, the amount of renally excreted C(f)P(-)-soman m rats should be sufficient to be of toxicological stgnificance if the agent could be reabsorbed from the bladder. The lethal effect brought about by mtravestcal admmutratton of C(f)P(f)-soman showed that this process can Indeed take place. Therefore, at first glance it might be suggested that the higher persistence in rats than in the two other species can be understood on the basts of the relatively high C(+)P(-)-soman levels found in urme and kidneys However, survival times of mtoxicated rats, whtch were immediately treated with the therapeuttc agent HI-6, stgmficantly decreased when accumulation of this C(&)P(-)-soman depot is prevented by rinsing and drainage of the bladder. Conversely, in rats intoxicated with 1 LD50 C(f)P(+)-soman and m PDP-pretreated rats intoxicated with 6 LD5o C(+)P(f)-soman and subsequently treated with HI-6, much higher urine levels of C(+)P(-)-soman were found than in similarly treated guinea pigs and marmosets, whereas these rats did not show late toxicity. Therefore, whatever the importance of a certam amount of C(+)P(-)-soman reabsorbed from the bladder for its persistence in rats, it is concluded that it does not explain late toxicity found for the agent in this species. References
7. 8. 9. 10. 11. 12. ::15:
O.L. WOLTHUIS, F. BERENDS and E. MEETER, Fundam Appl. Toxicol. 1. 183-192 (1981). H.P.M. VAN HELDEN and O.L. WOLTHUIS, Eur. J. Pharmacol. 89 271-274 (1983). H P BENSCHOP, F. BERENDS and L.P A. DE JONG, Fundam. Appl. Toxicol. 1 177182 (1981). H.P. BENSCHOP, E.C. BIJLEVELD, L P A. DE JONG, H.J. VAN DER WIEL and H.P.M. VAN HELDEN, Toxicol. Appl. Pharmacol B 490-500 (1987). H.P. BENSCHOP and L.P.A. DE JONG, Neurosci. Biobehav. Rev. 15.73-77 (1991). H.P.M. VAN HELDEN, H.J. VAN DER WIEL and 0 L. WOLTHUIS, J. Pharm. Pharmacol. a 35-41 (1988). H.P.M. VAN HELDEN, H.P. BENSCHOP and O.L. WOLTHUIS, J. Pharm. Pharmacol. 38 19-23 (1986). H.P. BENSCHOP, E.C. BIJLEVELD, M.F. OTTO, C.E A.M. DEGENHARDT, H.P.M. VAN HELDEN and L.P.A. DE JONG, Anal. Bmchem. 151242-253 (1985). M. ETO, J.E. CASIDA and T. ETO, B&hem. Pharmacol. e 337-352 (1962). D.M. MAXWELL, K.M. BRECHT and B.L. O’NEILL, Toxtcol. Lett. 29 35-42 (1987). D.E. LENZ, D.M. MAXWELL, R. PRATHER and L BALL, The Pharmacologist 25 111, (1983). E. HEILBRONN, I.-E. APPELGREN and A. SUNDWALL, Biochem. Pharmacol. ti 11891195 (1964). H.P. BENSCHOP and H.C. WESSELMAN, Arch. Toxicol. a 238-243 (1989). D. HANSEN, E. SCHAUM and 0. WASSERMAN, Arch.Toxicol. a 73-81 (1968). D. HANSEN, E. SCHAUM and 0. WASSERMAN, Biochem. Pharmacol. 11 1159-1162 (1968).