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Potentiation of Organophosphorus-Induced Delayed Neurotoxicity Following Phenyl Saligenin Phosphate Exposures in 2-, 5-, and 8-Week-Old Chickens
FUNDAMENTAL AND APPLIED TOXICOLOGY ARTICLE NO.
37, 64–70 (1997)
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Potentiation of Organophosphorus-Induced Delayed Neurotoxicity Following Phenyl Saligenin Phosphate Exposures in 2-, 5-, and 8-Week-Old Chickens Paul Harp,* Duke Tanaka, Jr.,† and Carey N. Pope* *Division of Toxicology, College of Pharmacy and Health Sciences, Northeast Louisiana University, Monroe, Louisiana 71209; and †Department of Anatomy, Michigan State University, East Lansing, Michigan 48824 Received June 24, 1996; accepted February 3, 1997
Certain organophosphorus compounds are capable of causing a delayed neurotoxicity that occurs weeks after the exposure. This syndrome is referred to as organophosphorusinduced delayed neurotoxicity (OPIDN) and is characterized by a distal ‘‘dying-back’’ or Wallerian-type degeneration of long axons in certain central and peripheral nerve tracts (Johnson, 1982). Clinical signs of the neuropathy in laboratory animals can range from slight incoordination to complete hindlimb paralysis. Initiation of OPIDN is generally agreed to be associated with a two-step process: phosphorylation (inhibition) of the protein neurotoxic esterase (NTE) and ‘‘aging’’ (i.e., loss of side chain group) of the phosphorylated enzyme (for reviews see Abou-Donia and Lapadula, 1990; Johnson, 1990; Richardson, 1992; Pope et al., 1993). Nonneuropathic NTE inhibitors bind the enzyme in a similar manner but the resulting complex is thought to be incapable of aging (Johnson, 1990). Nonneuropathic NTE inhibitors do not initiate OPIDN but can modify expression of the neuropathy induced by a neuropathic agent. In essence, pretreatment with the nonneuropathic agent provides protection from subsequent neuropathic insult (Johnson, 1970; Caroldi et al., 1984; Veronesi and Padilla, 1985) but posttreatment with the nonneuropathic compound potentiates the neuropathy (Pope and Padilla, 1990; Lotti et al., 1991; Pope et al., 1992). Prophylaxis by pretreatment with a nonneuropathic inhibitor is thought to be due to competitive binding: the NTE molecule is bound by the nonneuropathic agent and is therefore unavailable for interaction with the neuropathic organophosphorus compound (Johnson, 1974). It is unclear how posttreatment with the same compound could markedly amplify delayed neurotoxicity. OPIDN is usually studied in adult chickens: very young birds are completely resistant to clinical signs of OPIDN but not to the neural damage itself (Funk et al., 1994) and become more susceptible to the clinical signs as they mature (Bondy et al., 1960; Johnson and Barnes, 1970; Moretto et al., 1991; Pope et al., 1992; Peraica et al., 1993). Experimental use of phenyl saligenin phosphate (PSP), an analog of the neuropathic metabolite of tri-ortho-tolyl phosphate (TOTP),
Potentiation of Organophosphorus-Induced Delayed Neurotoxicity Following Phenyl Saligenin Phosphate Exposures in 2-, 5-, and 8-Week-Old Chickens. Harp, P., Tanaka, Jr., D., and Pope, C. N. (1997). Fundam. Appl. Toxicol. 37, 64–70. Phenylmethylsulfonyl fluoride (PMSF), a nonneuropathic inhibitor of neurotoxic esterase (NTE), is a known potentiator of organophosphorus-induced delayed neurotoxicity (OPIDN). The ability of PMSF posttreatment (90 mg/kg, sc, 4 hr after the last PSP injection) to modify development of delayed neurotoxicity was examined in 2-, 5-, and 8-week-old White Leghorn chickens treated either one, two, or three times (doses separated by 24 hr) with the neuropathic OP compound phenyl saligenin phosphate (PSP, 5 mg/kg, sc). NTE activity was measured in the cervical spinal cord 4 hr after the last PSP treatment. Development of delayed neurotoxicity was measured over a 16-day postexposure period. All PSP-treated groups exhibited ú97% NTE inhibition regardless of age or number of OP treatments. Twoweek-old birds did not develop clinical signs of neurotoxicity in response to either single or repeated OP treatment regimens nor following subsequent treatment with PMSF. Five-week-old birds were resistant to the clinical effects of a single PSP exposure and were minimally affected by repeated doses. PMSF posttreatment, however, significantly amplified the clinical effects of one, two, or three doses of PSP. A single exposure to PSP induced slight to moderate signs of delayed neurotoxicity in 8-week-old birds with more extensive neurotoxicity being noted following repeated dosing. As with 5-week-old birds, PMSF exacerbated the clinical signs of neurotoxicity when given after one, two, or three doses of PSP in 8-week-old birds. Axonal degeneration studies supported the clinical findings: PMSF posttreatment did not influence the degree of degeneration in 2-week-old chickens but resulted in more severe degeneration (relative to PSP only exposure) in cervical cords from both 5- and 8-week-old birds. The results indicate that PMSF does not alter the progression of delayed neurotoxicity in very young (2 weeks of age) chickens but potentiates PSP-induced delayed neurotoxicity in the presence of 0–3% residual NTE activity in older animals. We conclude that posttreatment with neuropathic or nonneuropathic NTE inhibitors, following virtually complete NTE inhibition by either single or repeated doses of a neuropathic agent in sensitive age groups, can modify both the clinical and morphological indices of delayed neurotoxicity. This study further supports the hypothesis that potentiation of OPIDN occurs through a mechanism unrelated to NTE. q 1997 Society of Toxicology.
0272-0590/97 $25.00 Copyright q 1997 by the Society of Toxicology. All rights of reproduction in any form reserved.
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provides certain advantages over other commonly used neuropathic OPs. Specifically, PSP does not require metabolic activation nor does it cause significant inhibition of acetylcholinesterase (Jortner and Ehrich, 1987). In the present study, the ability of the nonneuropathic NTE inhibitor phenylmethylsulfonyl fluoride to modify expression of neuropathy following single or repeated exposures to PSP was examined in three age groups (2, 5, and 8 weeks old) of young chickens. We hypothesized that phenylmethylsulfonyl fluoride (PMSF) posttreatment would potentiate delayed neurotoxicity following either single or multiple doses of PSP sufficient to inhibit essentially all NTE activity. MATERIALS AND METHODS Materials. Mipafox (N, N *-diisopropyl-diamido-phosphoro fluoridate, purity ú98%) was purchased from Chemsyn (Lenexa, KS). Paraoxon (O, O*-diethyl-p-nitrophenyl phosphate) was purchased from Aldrich Chemical Company (Milwaukee, WI). Aliquots of paraoxon were further purified prior to assay (Johnson, 1977). Phenyl valerate was synthesized essentially as described by Johnson (1977) and distilled (bp Å 70–70.57C, 0.5 mm Hg). Phenylmethylsulfonyl fluoride (purity ú99%) was purchased from Sigma Chemical Company (St. Louis, MO). Cyclic phenyl saligenin phosphate (purity, 97%, as determined by GC-MS) was a generous gift from Dr. Marion Ehrich and was synthesized as described by Jortner and Ehrich (1987). All other chemicals were reagent grade. Animals and treatments. White Leghorn pullets (C.M. Estes Hatchery, Springfield, MO) were obtained at 1 day of age and maintained at 257C under a 12-hr light:dark illumination cycle with food (Brood’s Best Chick Starter/Grower Crumbles, Mountaire Feeds Inc., North Little Rock, AR) and water available ad libitum. Separate groups of 2-, 5-, and 8-week-old chickens were treated zero, one, two, or three times (treatments separated by 24 hr) with PSP (5 mg/kg, sc, 10 mg/ml in dimethyl sulfoxide, DMSO). Control birds received DMSO only (0.5 ml/kg, sc). Four hours after the last PSP injection the birds were either euthanized and their cervical cords were dissected for NTE determination or they were treated with either glycerol formal (GF, 1 ml/kg, sc) or PMSF (90 mg/kg, sc, 90 mg/ml in GF) and observed for clinical signs of OPIDN over a 16-day period. After observation on Day 16, birds were euthanized by decapitation and the cervical cords were collected in 10% neutral buffered formalin for morphologic analysis. Neurotoxic esterase assay. Cervical cords were removed and stored at 0707C until time of assay. NTE activity was measured by the method of Johnson (1977) as modified by Pope and Padilla (1989). Briefly, tissues were thawed and weighed, and homogenates (1:10, w/v) were prepared in 50 mM tris[hydroxymethyl]aminomethane buffer (pH 8.0 @ 257C) containing 0.2 mM EDTA (Tris–EDTA). Homogenates were centrifuged (9000g for 15 min) and the resulting supernatants were used for NTE determination. Reaction volumes of paired samples contained 50 ml homogenate, 40 mM paraoxon, and 5 ml phenyl valerate (PV, 25 mg/ml in dimethyl formamide) with or without 50 mM mipafox. Tubes containing homogenate and OPs (or vehicle) were preincubated for 20 min at 377C. Phenyl valerate was then added for a 20-min incubation (377C). The reaction was stopped by addition of 500 ml of Tris–EDTA buffer containing 1% sodium dodecyl sulfate and 0.025% 4-aminoantipyrine. NTE activity was determined as the difference in hydrolysis of PV in the presence and absence of mipafox. Protein content was measured according to the method of Lowry and coworkers (1951) and NTE activity was reported as nanomoles of PV hydrolyzed/minute/milligram of protein. Clinical signs of neurotoxicity. All birds were coded on Day 0 such that the observations were performed ‘‘blind.’’ Clinical signs of OPIDN (n Å 4–5 birds/group) were measured at 6, 8, 10, 12, 14, and 16 days after
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PMSF according to the following scale (Roberts et al., 1983). In brief: 0, no signs; 1/2, slight, indefinite signs; 3/4, moderate degree of incoordination during movement; 5/6, severe impairment of ability to stand and walk; 7/ 8, virtual to complete hindlimb paralysis. Morphologic analysis. Chickens (n Å 4–5 birds/group) were euthanized by decapitation after observation on Day 16. The cervical region of the spinal cord was carefully removed and stored in a 10% neutral buffered formalin solution prior to processing. Serial sections through the C1 cervical segment were cut in the frontal plane at a thickness of 40 mm using a freezing microtome. A variation of the Fink–Heimer silver impregnation method for selectively staining degenerating axons and terminals (Tanaka, 1976) was used to analyze every fifth section. This method has been used in several previous studies to assess the neuropathological effects of different organophosphorus compounds on the central nervous system in birds and has been shown to be very sensitive in identifying organophosphorus-induced axonal degeneration (Tanaka and Bursian, 1989; Tanaka et al., 1990, 1992a,b; Pope et al., 1992; Varghese et al., 1995). The gracile fasciculus and dorsal spinocerebellar tracts were examined microscopically at the level of the first spinal cervical segment for the presence of silver-impregnated axonal degeneration. Sections for examination were selected far enough from the site of the decapitation so that traumatic indicators of decapitation (extravasated blood, torn or twisted fiber tracts, irregular or torn spinal cord borders) were not present. The gracile fasciculus and spinocerebellar tract were each ranked separately and assigned degeneration scores according to the following scale: 0, no degeneration; 1, small numbers of scattered degenerating axons with the majority of the tract free of degeneration; 2, moderate numbers of degenerating fibers extending over a larger area of the affected tract; 3, large numbers of degenerating axons present throughout the affected tract. Tissue samples were coded such that the analyses were performed blind. Data analysis. Enzyme data were reported as means { SE and were analyzed by a one-way analysis of variance followed by Student–Newman– Keuls post hoc comparison. Ataxia data were reported as median { interquartile ranges and were tested for significance at each timepoint with the Mann–Whitney Rank Sum test. Morphologic data were reported as median scores. In all comparisons p õ 0.05 was taken to indicate statistical significance. All analyses were performed using the SigmaStat Statistical Analysis System software package (Jandel Scientific, San Rafael, CA).
RESULTS
Inhibition of Neurotoxic Esterase Activity Single doses of PSP (5 mg/kg, sc) caused ú97% inhibition of cervical cord NTE 4 hr after treatment in all age groups (Table 1). No additional significant reduction was seen with multiple OP exposures. Extensive NTE inhibition (ú95%) was also noted in birds treated with a single dose of PMSF (90 mg/kg, sc). The different treatment schedules caused similar reductions of enzyme activity across the different age groups. Clinical Signs of OPIDN No clinical signs of OPIDN were observed in any of the 2-week-old birds regardless of the number of PSP treatments or PMSF posttreatment (data not shown). Five-week-old birds (Figs. 1A–1D) were also resistant to OPIDN after receiving only PSP, but PMSF posttreatment was associated with moderate to severe clinical neurotoxicity in all three PSP-treated groups. Single doses of PSP caused minor signs
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FIG. 1. Development of clinical signs of neuropathy in 5-week-old chickens. (A) Ataxia in control birds and birds treated with PMSF (90 mg/kg, sc) only. (B) Compares a single dose of PSP (5 mg/kg, sc) to a single dose of PSP followed 4 hr later by PMSF. (C and D) Compare two or three doses of PSP only to two or three doses of PSP followed by PMSF. Data are shown as median ataxia scores { interquartile ranges. Asterisks indicate significant effects of PMSF posttreatment (i.e., potentiation).
of clinical dysfunction in 8-week-old birds (Figs. 2A–2D) with multiple treatments causing more severe effects than a single dose. PMSF exacerbated delayed neurotoxicity in all 8-week-old birds receiving one, two, or three doses of PSP.
spinocerebellar tracts of the cervical spinal cord. In both tracts the silver-impregnated degeneration appeared as fragmented, irregularly arranged, black silver-impregnated fibers on a light brown or yellow background. The density of the degeneration was dependent on the particular treatment to which the chick was exposed (Fig. 3). No axonal degeneration was found in the cervical cords from any control birds (n Å 4–5 birds/age group; data not shown). Cords from 2-
Morphology Degeneration induced by exposure to PSP appeared consistently and selectively in the gracile fasciculus and dorsal
TABLE 1 Inhibition of Neurotoxic Esterase Activity in Cervical Cords of 2-, 5-, and 8-Week-Old Chickens Treated with Single Doses of PMSF (90 mg/kg, sc) or Single or Repeated Doses of PSP (5 mg/kg, sc) 2-week-old birds
Treatment
n
nmol/min/mg protein
Control PMSF PSP 1 1 PSP 1 2 PSP 1 3
4 4 4 4 3
5.00 0.13 0.14 0.04 0.10
{ { { { {
0.74 0.07 0.02 0.05 0.14
5-week-old birds
% control
n
nmol/min/mg protein
{ { { { {
6 6 6 5 5
9.14 0.30 0.15 00.07 00.15
100.00 2.70 2.88 0.84 1.99
14.87 1.35* 0.42* 0.95* 2.73*
{ { { { {
0.45 0.04 0.05 0.06 0.11
8-week-old birds
% control 100.00 3.29 1.66 00.79 01.68
{ { { { {
4.87 0.47* 0.52* 0.62* 1.26*
n
nmol/min/mg protein
6 4 6 6 6
9.86 0.34 0.11 0.18 0.19
{ { { { {
Note. Data are expressed as means { SE. * Statistically significant at p õ 0.05 by one-way analysis of variance and Student–Newman–Keuls multiple comparison.
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0.38 0.10 0.02 0.04 0.05
% control 100.00 3.48 1.24 1.83 1.90
{ { { { {
3.85 1.04* 0.21* 0.42* 0.49*
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FIG. 2. Development of clinical signs of neuropathy in 8-week-old chickens. (A) Ataxia in control birds and birds treated with PMSF (90 mg/kg, sc) only. (B) Compares a single dose of PSP (5 mg/kg, sc) to a single dose of PSP followed 4 hr later by PMSF. (C and D) Compare two or three doses of PSP only to two or three doses of PSP followed by PMSF. Data are shown as median ataxia scores { interquartile ranges. Asterisks indicate significant effects of PMSF posttreatment. Pound signs indicate significant effects of repeated doses of PSP compared to a single dose.
week-old birds had only small amounts of axonal degeneration even following multiple PSP treatments; PMSF posttreatment did not increase degeneration in this age group (Fig. 4A). In the gracile fasciculi from 5-week-old birds, moderate amounts of axonal degeneration were present after single or repeated PSP exposures (Fig. 4B). More extreme (moderate to heavy) degeneration was found in the gracile fasciculi of birds that received both PSP and PMSF. In the spinocerebellar tracts of 5-week-old birds, degeneration was light to moderate in PSP-only treated birds but moderate to heavy in PSP/PMSF-treated birds. Between the two regions, the gracile fasciculus was more severely affected than the spinocerebellar tracts by PSP-only exposures. PMSF posttreatment increased the severity of damage in both regions, with somewhat greater changes (relative to PSP-only) noted in the spinocerebellar tracts. In the gracile fasciculi of 8-week-old birds, degeneration scores ranged from moderate to heavy depending on the number of PSP exposures, with little difference between the PSP-only groups and their respective PMSF posttreated groups (Fig. 4C). In contrast, the spinocerebellar tracts from 8-week-old birds showed light to moderate degeneration in response to PSP only but moderate to heavy degeneration after PSP followed by PMSF.
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DISCUSSION
Inhibition of NTE is the putative first step in initiation of OPIDN (for reviews see Abou-Donia and Lapadula, 1990; Johnson, 1990; Lotti, 1992; Richardson, 1992). In the present studies, single doses of phenyl saligenin phosphate inhibited NTE activity in cervical spinal cord by at least 97%. Subsequent doses of PSP did not significantly increase enzyme inhibition. In contrast to a report by Moretto and co-workers (1991), we found that spinal cord NTE activity in control birds increased with age (5.00 { 0.74, 9.14 { 0.45, and 9.86 { 0.38 nmol/min/mg protein for 2-, 5-, and 8-week-old birds, respectively). Differences in tissue (cervical vs lumbosacral regions) or assay procedures could account for these contrasting results. Two-week-old birds were completely resistant to clinical signs of OPIDN following either single or repeated doses of PSP and exhibited only slight degrees of axonal degeneration, even after PMSF posttreatment. Five-week-old birds were relatively resistant to clinical signs of OPIDN after one, two, or three doses of PSP but developed extensive deficits in locomotor function with PMSF posttreatment in all cases. In general, the degree of axonal degeneration correlated with clinical dysfunction. Similar to previous studies
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using diisopropyl phosphorofluoridate (DFP) as the neuropathic agent (Pope et al., 1993), 8-week-old birds exhibited minimal degrees of clinical dysfunction following a single neuropathic OP exposure. Multiple PSP exposures induced more extensive clinical deficits, however. In addition, PMSF posttreatment amplified the delayed neurotoxicity in 8-weekold chickens initiated by one, two, or three doses of PSP. In 5-week-old chickens, repeated PSP treatments had little effect on the development of clinical signs of neurotoxicity. These findings agree with a previous study (Pope et al., 1993) reporting minimal effects of repeated DFP exposures. In contrast, relatively similar ataxia scores were noted in 8week-old birds treated with either (a) a single dose of PSP followed by PMSF or (b) two doses of PSP. These findings suggest that a subsequent exposure to PSP may be capable of potentiating the neurotoxic effects of a prior dose, but only in more mature animals. Collectively, these studies suggest that some neuropathic OPs may have multiple target sites of relevance to OPIDN and may be capable of both initiation and potentiation of delayed neurotoxicity. The resistance to clinical dysfunction following either repeated PSP or PMSF posttreatment in 2-week-old chickens, the resistance to potentiation by repeated OP exposures but sensitivity to potentiation of both clinical and morphological indicators of OPIDN by PMSF posttreatment in 5-week-old chickens, and finally, the greater sensitivity to both repeated OP exposures and PMSF posttreatment in 8-week-old chickens suggest that an age-related gradient exists in the degree of sensitivity to both initiators and potentiators of delayed neurotoxicity. The results of this study indicate that both the gracile fasciculus and the dorsal spinocerebellar tracts undergo distal axonal degeneration following exposure to PSP. These data are consistent with previous reports indicating that both of these tracts are susceptible to a variety of organophosphorus compounds (Tanaka and Bursian, 1989; Tanaka et al., 1990; Carboni et al., 1992; Dyer et al., 1992). Although both dorsal spinocerebellar and ventral spinocerebellar tracts have been reported in birds (Whitlock, 1952; Karten and Hodos, 1967), the present results indicate that the larger dorsal tract undergoes more severe degeneration after exposure to PSP and PSP/PMSF, a finding similar to that reported in chickens after exposure to tri-ortho-tolyl phosphate (Tanaka and Bursian, 1989) and diisopropylphosphorofluoridate (Tanaka et al., 1990). Previous studies have hypothesized that degeneration of the dorsal spinocerebellar tract, which has been shown in chickens to terminate as mossy fiber afferents in the cerebellar cortex (Okado et al., 1987), may be closely linked to the onset of ataxia (Pope et al., 1993; Funk et al., 1994). The morphological data in the present FIG. 3. Photomicrographs illustrating the different degeneration scores (0–3) noted in the dorsal spinocerebellar tract following administration of PSP and/or PMSF in 8-week-old chickens. (A) The absence of degeneration (degeneration score Å 0) following an injection of PMSF only. (B) The light punctate degeneration (degeneration score Å 1) present after a single injec-
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tion of PSP. (C and D) Illustrate moderate (degeneration score Å 2) to heavy (degeneration score Å 3) degeneration present after injections of both PSP and PMSF. Arrowheads indicate degenerating axons. Scale bar, 50 mm.
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exist. For example, cords from 2-week-old birds had slight amounts of degeneration but the birds were completely resistant to clinical dysfunction. These results agree with a previous study (Funk et al., 1994) following repeated DFP exposures in 2-week-old chickens. Comparison of similarly treated groups of 5- and 8-week-old birds (Figs. 4B and 4C) indicated very similar degrees of axonal damage but the older birds exhibited more severe clinical signs of delayed neurotoxicity. These observations may indicate that younger animals are not just resistant to the initial neuropathic insult but, because of the developing state of their nervous system, are also more successful at compensating for the damage. Although the putative target for initiation of OPIDN is neurotoxic esterase, Pope and Padilla (1990) proposed that potentiation of OPIDN by nonneuropathic NTE inhibitors did not involve NTE. The present study supports this hypothesis by demonstrating that PMSF can amplify clinical and morphological indicators of OPIDN in 5-week-old chickens when given after one, two, or three injections of PSP; dosing regimens which cause from 97 to 100% inhibition of NTE. In 8-week-old birds, PMSF posttreatment also exacerbated clinical dysfunction following similar dosing regimens. In addition, a second dose of PSP markedly increased the delayed neurotoxicity of an initial dose of the OP. More recently, Moretto and co-workers (1994) demonstrated potentiation of OPIDN by O-(2-chloro-2,3,3-trifluorocyclobutyl) Oethyl S-propyl ester (KBR-2822) at doses which did not significantly affect NTE activity. Collectively, these studies support the hypothesis that another target molecule is involved in potentiation of OPIDN. In conclusion, we have demonstrated that PMSF can potentiate the delayed neurotoxic effects of PSP in the presence of less than 3% residual NTE activity. The results further support the hypothesis that potentiation of OPIDN involves a target molecule other than NTE. FIG. 4. Axonal degeneration in the dorsal columns (shaded bars) and spinocerebellar tracts (striped bars) of cervical cords from 2-, 5-, and 8-weekold chickens treated with PSP (5 mg/kg, sc) with or without PMSF (90 mg/ kg, sc). Tissues were collected 16 days after treatment and examined for degeneration using a modification of the Fink–Heimer silver staining technique. Morphological effects in 2-week-old chickens (A) were measured in birds receiving either three doses of PSP or three doses of PSP followed by PMSF 4 hr after the final treatment. Axonal degeneration was examined in 5- (B) and 8- (C) week-old birds following one, two, or three doses of PSP with or without PMSF posttreatment. No axonal degeneration was noted in any samples from any age group treated with vehicles or PMSF alone.
study add to the results from these previous investigations by showing that although PMSF posttreatment increases degeneration in both regions of the cervical cord, it appears to produce greater changes (relative to OP exposure only) in the dorsal spinocerebellar tract. While the morphological data in the present study generally support the clinical findings, some interesting contrasts
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ACKNOWLEDGMENTS The authors appreciate the technical assistance of Mr. Thuc Pham and Dr. Tamal Chakraborti. This research was partially supported through cooperative agreement CR820229-01 between the United States Environmental Protection Agency and Northeast Louisiana University (C.N.P.).
REFERENCES Abou-Donia, M. B., and Lapadula, D. M. (1990). Mechanisms of organophosphorus ester-induced delayed neurotoxicity: Type I and Type II. Annu. Rev. Pharmacol. Toxicol. 30, 405–440. Bondy, H. F., Field, E. J., Worden, A. N., and Hughes, J. P. W. (1960). A study on the acute toxicity of the tri-aryl phosphates used as plasticizers. Br. J. Ind. Med. 17, 190–200. Carboni, D., Ehrich, M., Dyer, K., and Jortner, B. S. (1992). Comparative evolution of mipafox-induced delayed neuropathy in rats and hens. Neurotoxicology 13, 723–734. Caroldi, S., Lotti, M., and Masutti, A. (1984). Intraarterial injection of diisopropylfluorophosphate or phenylmethane sulfonyl fluoride produces
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unilateral neuropathy or protection, respectively, in hens. Biochem. Pharmacol. 33, 3213–3217. Dyer, K., Jortner, B. S., Shell, L. G., and Ehrich, M. (1992). Comparative dose–response studies of organophosphorus ester-induced delayed neuropathy in rats and hens administered mipafox. Neurotoxicology 13, 745– 756. Funk, K. A., Henderson, J. D., Liu, C. H., Higgins, R. J., and Wilson, B. W. (1994). Neuropathology of organophosphate-induced delayed neuropathy (OPIDN) in young chicks. Arch. Toxicol. 68, 308–316. Johnson, M. K. (1970). Organophosphorus and other inhibitors of brain ‘‘neurotoxic esterase’’ and the development of delayed neurotoxicity in hens. Biochem. J. 120, 523–531. Johnson, M. K., and Barnes, J. M. (1970). Age and the sensitivity of chicks to the delayed neurotoxic effects of some organophosphorus compounds. Biochem. Pharmacol. 19, 3045–3047. Johnson, M. K. (1974). The primary biochemical lesion leading to the delayed neurotoxic effects of some organophosphorus esters. J. Neurochem. 23, 785–789. Johnson, M. K. (1977). Improved assay of neurotoxic esterase for screening organophosphates for delayed neurotoxicity potential. Arch. Toxicol. 37, 113–115. Johnson, M. K. (1982). The target for initiation of delayed neurotoxicity by organophosphorus esters: Biochemical studies and toxicological applications. In Reviews in Biochemical Toxicology (E. Hodgson, J. R. Bend, and R. M. Philpot, Eds.), Vol. 4, pp. 141–212. Elsevier, New York. Johnson, M. K. (1990). Organophosphates and delayed neuropathy—Is NTE alive and well? Toxicol. Appl. Pharmacol. 102, 385–399. Jortner, B. S., and Ehrich, M. (1987). Neuropathological effects of phenyl saligenin phosphate in chickens. Neurotoxicology 8, 303–314. Karten, H. J., and Hodos, W. (1967). A Stereotaxic Atlas of the Brain of the Pigeon (Columbia livia), 193 pp. Johns Hopkins Press, Baltimore. Lotti, M. (1992). The pathogenesis of organophosphate polyneuropathy. Crit. Rev. Toxicol. 21, 465–487. Lotti, M., Caroldi, S., Capodicasa, E., and Moretto, A. (1991). Promotion of organophosphate-induced delayed polyneuropathy by phenylmethanesulfonyl fluoride. Toxicol. Appl. Pharmacol. 108, 234–241. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Moretto, A., Capodicasa, E., Peraica, M., and Lotti, M. (1991). Age sensitivity to organophosphate-induced delayed polyneuropathy. Biochem. Pharmacol. 41, 1497–1504. Moretto, A., Bertolazzi, M., and Lotti, M. (1994). The phosphorothioic acid O-(2-chloro-2,3,3-trifluorocyclobutyl) O-ethyl S-propyl ester exacerbates organophosphate polyneuropathy without inhibition of neuropathy target esterase. Toxicol. Appl. Pharmacol. 129, 133–137. Okado, N., Ito, R., and Homma, S. (1987). The terminal distribution pattern of spinocerebellar fibers: An anterograde labelling study in the posthatching chick. Anat. Embryol. 176, 175–182.
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Peraica, M., Capodicasa, E., Moretto, A., and Lotti, M. (1993). Organophosphate polyneuropathy in chicks. Biochem. Pharmacol. 45, 131–135. Pope, C. N., and Padilla, S. S. (1989). Chromatographic characterization of neurotoxic esterase. Biochem. Pharmacol. 38, 181–188. Pope, C. N., and Padilla, S. (1990). Potentiation of organophosphorus-induced delayed neurotoxicity by phenylmethylsulfonyl fluoride. J. Toxicol. Environ. Health 31, 261–273. Pope, C. N., Chapman, M. L., Tanaka, D., Jr., and Padilla, S. (1992). Phenylmethylsulfonyl fluoride alters sensitivity to organophosphorus-induced delayed neurotoxicity in developing animals. Neurotoxicology 13, 355– 364. Pope, C. N., Tanaka, D., Jr., and Padilla, S. (1993). The role of neurotoxic esterase (NTE) in the prevention and potentiation of organophosphorusinduced delayed neurotoxicity (OPIDN). Chem.-Biol. Interact. 87, 395– 406. Richardson, R. J. (1992). Interactions of organophosphorus compounds with neurotoxic esterase. In Organophosphates: Chemistry, Fate, and Effects (J. E. Chambers and P. E. Levi, Eds.), pp. 299–323. Academic Press, San Diego. Roberts, N. L., Fairley, C., and Phillips, C. (1983). Screening, acute delayed and subchronic neurotoxicity studies in the hen: Measurements and evaluations of clinical signs following administration of TOCP. Neurotoxicology 4, 263–270. Tanaka, D., Jr. (1976). Thalamic projections of the dorsomedial prefrontal cortex in the Rhesus monkey (Macaca mulatta). Brain Res. 110, 21–38. Tanaka, D., Jr., and Bursian, S. J. (1989). Degeneration patterns in the chicken central nervous system induced by ingestion of the organophosphorus delayed neurotoxin tri-ortho-tolyl phosphate: A silver impregnation study. Brain Res. 484, 240–256. Tanaka, D., Jr., Bursian, S. J., and Lehning, E. (1990). Selective axonal and terminal degeneration in the chicken brainstem and cerebellum following exposure to bis(1-methylethyl) phosphorofluoridate (DFP). Brain Res. 519, 200–208. Tanaka, D., Jr., Bursian, S. J., and Lehning, E. J. (1992a). Neuropathological effects of triphenyl phosphite in the central nervous system of the hen (Gallus domesticus). Fundam. Appl. Toxicol. 18, 72–78. Tanaka, D., Jr., Bursian, S. J., and Lehning, E. J. (1992b). Silver impregnation of organophosphorus-induced delayed neuropathy in the central nervous system. In The Vulnerable Brain and Environmental Risks. Vol. 2. Toxins in Food (R. Isaacson and K. Jensen, Eds.), pp. 215–234. Plenum, New York. Varghese, R. G., Bursian, S. J., Tobias, C., and Tanaka, D., Jr. (1995). Organophosphorus-induced delayed neurotoxicity: A comparative study of the effects of tri-ortho-tolyl phosphate and triphenyl phosphite on the central nervous system of the Japanese quail. Neurotoxicology 16, 45– 54. Veronesi, B., and Padilla, S. (1985). Phenylmethylsulfonyl fluoride protects rats from mipafox-induced delayed neuropathy. Toxicol. Appl. Pharmacol. 81, 258–264. Whitlock, D. G. (1952). A neurohistological and neurophysiological study of afferent fiber tracts and receptive areas of the avian cerebellum. J. Comp. Neurol. 97, 567–635.
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Potentiation of Organophosphorus-Induced Delayed Neurotoxicity Following Phenyl Saligenin Phosphate Exposures in 2-, 5-, and 8-Week-Old Chickens Paul Harp,* Duke Tanaka, Jr.,† and Carey N. Pope* *Division of Toxicology, College of Pharmacy and Health Sciences, Northeast Louisiana University, Monroe, Louisiana 71209; and †Department of Anatomy, Michigan State University, East Lansing, Michigan 48824 Received June 24, 1996; accepted February 3, 1997
Certain organophosphorus compounds are capable of causing a delayed neurotoxicity that occurs weeks after the exposure. This syndrome is referred to as organophosphorusinduced delayed neurotoxicity (OPIDN) and is characterized by a distal ‘‘dying-back’’ or Wallerian-type degeneration of long axons in certain central and peripheral nerve tracts (Johnson, 1982). Clinical signs of the neuropathy in laboratory animals can range from slight incoordination to complete hindlimb paralysis. Initiation of OPIDN is generally agreed to be associated with a two-step process: phosphorylation (inhibition) of the protein neurotoxic esterase (NTE) and ‘‘aging’’ (i.e., loss of side chain group) of the phosphorylated enzyme (for reviews see Abou-Donia and Lapadula, 1990; Johnson, 1990; Richardson, 1992; Pope et al., 1993). Nonneuropathic NTE inhibitors bind the enzyme in a similar manner but the resulting complex is thought to be incapable of aging (Johnson, 1990). Nonneuropathic NTE inhibitors do not initiate OPIDN but can modify expression of the neuropathy induced by a neuropathic agent. In essence, pretreatment with the nonneuropathic agent provides protection from subsequent neuropathic insult (Johnson, 1970; Caroldi et al., 1984; Veronesi and Padilla, 1985) but posttreatment with the nonneuropathic compound potentiates the neuropathy (Pope and Padilla, 1990; Lotti et al., 1991; Pope et al., 1992). Prophylaxis by pretreatment with a nonneuropathic inhibitor is thought to be due to competitive binding: the NTE molecule is bound by the nonneuropathic agent and is therefore unavailable for interaction with the neuropathic organophosphorus compound (Johnson, 1974). It is unclear how posttreatment with the same compound could markedly amplify delayed neurotoxicity. OPIDN is usually studied in adult chickens: very young birds are completely resistant to clinical signs of OPIDN but not to the neural damage itself (Funk et al., 1994) and become more susceptible to the clinical signs as they mature (Bondy et al., 1960; Johnson and Barnes, 1970; Moretto et al., 1991; Pope et al., 1992; Peraica et al., 1993). Experimental use of phenyl saligenin phosphate (PSP), an analog of the neuropathic metabolite of tri-ortho-tolyl phosphate (TOTP),
Potentiation of Organophosphorus-Induced Delayed Neurotoxicity Following Phenyl Saligenin Phosphate Exposures in 2-, 5-, and 8-Week-Old Chickens. Harp, P., Tanaka, Jr., D., and Pope, C. N. (1997). Fundam. Appl. Toxicol. 37, 64–70. Phenylmethylsulfonyl fluoride (PMSF), a nonneuropathic inhibitor of neurotoxic esterase (NTE), is a known potentiator of organophosphorus-induced delayed neurotoxicity (OPIDN). The ability of PMSF posttreatment (90 mg/kg, sc, 4 hr after the last PSP injection) to modify development of delayed neurotoxicity was examined in 2-, 5-, and 8-week-old White Leghorn chickens treated either one, two, or three times (doses separated by 24 hr) with the neuropathic OP compound phenyl saligenin phosphate (PSP, 5 mg/kg, sc). NTE activity was measured in the cervical spinal cord 4 hr after the last PSP treatment. Development of delayed neurotoxicity was measured over a 16-day postexposure period. All PSP-treated groups exhibited ú97% NTE inhibition regardless of age or number of OP treatments. Twoweek-old birds did not develop clinical signs of neurotoxicity in response to either single or repeated OP treatment regimens nor following subsequent treatment with PMSF. Five-week-old birds were resistant to the clinical effects of a single PSP exposure and were minimally affected by repeated doses. PMSF posttreatment, however, significantly amplified the clinical effects of one, two, or three doses of PSP. A single exposure to PSP induced slight to moderate signs of delayed neurotoxicity in 8-week-old birds with more extensive neurotoxicity being noted following repeated dosing. As with 5-week-old birds, PMSF exacerbated the clinical signs of neurotoxicity when given after one, two, or three doses of PSP in 8-week-old birds. Axonal degeneration studies supported the clinical findings: PMSF posttreatment did not influence the degree of degeneration in 2-week-old chickens but resulted in more severe degeneration (relative to PSP only exposure) in cervical cords from both 5- and 8-week-old birds. The results indicate that PMSF does not alter the progression of delayed neurotoxicity in very young (2 weeks of age) chickens but potentiates PSP-induced delayed neurotoxicity in the presence of 0–3% residual NTE activity in older animals. We conclude that posttreatment with neuropathic or nonneuropathic NTE inhibitors, following virtually complete NTE inhibition by either single or repeated doses of a neuropathic agent in sensitive age groups, can modify both the clinical and morphological indices of delayed neurotoxicity. This study further supports the hypothesis that potentiation of OPIDN occurs through a mechanism unrelated to NTE. q 1997 Society of Toxicology.
0272-0590/97 $25.00 Copyright q 1997 by the Society of Toxicology. All rights of reproduction in any form reserved.
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provides certain advantages over other commonly used neuropathic OPs. Specifically, PSP does not require metabolic activation nor does it cause significant inhibition of acetylcholinesterase (Jortner and Ehrich, 1987). In the present study, the ability of the nonneuropathic NTE inhibitor phenylmethylsulfonyl fluoride to modify expression of neuropathy following single or repeated exposures to PSP was examined in three age groups (2, 5, and 8 weeks old) of young chickens. We hypothesized that phenylmethylsulfonyl fluoride (PMSF) posttreatment would potentiate delayed neurotoxicity following either single or multiple doses of PSP sufficient to inhibit essentially all NTE activity. MATERIALS AND METHODS Materials. Mipafox (N, N *-diisopropyl-diamido-phosphoro fluoridate, purity ú98%) was purchased from Chemsyn (Lenexa, KS). Paraoxon (O, O*-diethyl-p-nitrophenyl phosphate) was purchased from Aldrich Chemical Company (Milwaukee, WI). Aliquots of paraoxon were further purified prior to assay (Johnson, 1977). Phenyl valerate was synthesized essentially as described by Johnson (1977) and distilled (bp Å 70–70.57C, 0.5 mm Hg). Phenylmethylsulfonyl fluoride (purity ú99%) was purchased from Sigma Chemical Company (St. Louis, MO). Cyclic phenyl saligenin phosphate (purity, 97%, as determined by GC-MS) was a generous gift from Dr. Marion Ehrich and was synthesized as described by Jortner and Ehrich (1987). All other chemicals were reagent grade. Animals and treatments. White Leghorn pullets (C.M. Estes Hatchery, Springfield, MO) were obtained at 1 day of age and maintained at 257C under a 12-hr light:dark illumination cycle with food (Brood’s Best Chick Starter/Grower Crumbles, Mountaire Feeds Inc., North Little Rock, AR) and water available ad libitum. Separate groups of 2-, 5-, and 8-week-old chickens were treated zero, one, two, or three times (treatments separated by 24 hr) with PSP (5 mg/kg, sc, 10 mg/ml in dimethyl sulfoxide, DMSO). Control birds received DMSO only (0.5 ml/kg, sc). Four hours after the last PSP injection the birds were either euthanized and their cervical cords were dissected for NTE determination or they were treated with either glycerol formal (GF, 1 ml/kg, sc) or PMSF (90 mg/kg, sc, 90 mg/ml in GF) and observed for clinical signs of OPIDN over a 16-day period. After observation on Day 16, birds were euthanized by decapitation and the cervical cords were collected in 10% neutral buffered formalin for morphologic analysis. Neurotoxic esterase assay. Cervical cords were removed and stored at 0707C until time of assay. NTE activity was measured by the method of Johnson (1977) as modified by Pope and Padilla (1989). Briefly, tissues were thawed and weighed, and homogenates (1:10, w/v) were prepared in 50 mM tris[hydroxymethyl]aminomethane buffer (pH 8.0 @ 257C) containing 0.2 mM EDTA (Tris–EDTA). Homogenates were centrifuged (9000g for 15 min) and the resulting supernatants were used for NTE determination. Reaction volumes of paired samples contained 50 ml homogenate, 40 mM paraoxon, and 5 ml phenyl valerate (PV, 25 mg/ml in dimethyl formamide) with or without 50 mM mipafox. Tubes containing homogenate and OPs (or vehicle) were preincubated for 20 min at 377C. Phenyl valerate was then added for a 20-min incubation (377C). The reaction was stopped by addition of 500 ml of Tris–EDTA buffer containing 1% sodium dodecyl sulfate and 0.025% 4-aminoantipyrine. NTE activity was determined as the difference in hydrolysis of PV in the presence and absence of mipafox. Protein content was measured according to the method of Lowry and coworkers (1951) and NTE activity was reported as nanomoles of PV hydrolyzed/minute/milligram of protein. Clinical signs of neurotoxicity. All birds were coded on Day 0 such that the observations were performed ‘‘blind.’’ Clinical signs of OPIDN (n Å 4–5 birds/group) were measured at 6, 8, 10, 12, 14, and 16 days after
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PMSF according to the following scale (Roberts et al., 1983). In brief: 0, no signs; 1/2, slight, indefinite signs; 3/4, moderate degree of incoordination during movement; 5/6, severe impairment of ability to stand and walk; 7/ 8, virtual to complete hindlimb paralysis. Morphologic analysis. Chickens (n Å 4–5 birds/group) were euthanized by decapitation after observation on Day 16. The cervical region of the spinal cord was carefully removed and stored in a 10% neutral buffered formalin solution prior to processing. Serial sections through the C1 cervical segment were cut in the frontal plane at a thickness of 40 mm using a freezing microtome. A variation of the Fink–Heimer silver impregnation method for selectively staining degenerating axons and terminals (Tanaka, 1976) was used to analyze every fifth section. This method has been used in several previous studies to assess the neuropathological effects of different organophosphorus compounds on the central nervous system in birds and has been shown to be very sensitive in identifying organophosphorus-induced axonal degeneration (Tanaka and Bursian, 1989; Tanaka et al., 1990, 1992a,b; Pope et al., 1992; Varghese et al., 1995). The gracile fasciculus and dorsal spinocerebellar tracts were examined microscopically at the level of the first spinal cervical segment for the presence of silver-impregnated axonal degeneration. Sections for examination were selected far enough from the site of the decapitation so that traumatic indicators of decapitation (extravasated blood, torn or twisted fiber tracts, irregular or torn spinal cord borders) were not present. The gracile fasciculus and spinocerebellar tract were each ranked separately and assigned degeneration scores according to the following scale: 0, no degeneration; 1, small numbers of scattered degenerating axons with the majority of the tract free of degeneration; 2, moderate numbers of degenerating fibers extending over a larger area of the affected tract; 3, large numbers of degenerating axons present throughout the affected tract. Tissue samples were coded such that the analyses were performed blind. Data analysis. Enzyme data were reported as means { SE and were analyzed by a one-way analysis of variance followed by Student–Newman– Keuls post hoc comparison. Ataxia data were reported as median { interquartile ranges and were tested for significance at each timepoint with the Mann–Whitney Rank Sum test. Morphologic data were reported as median scores. In all comparisons p õ 0.05 was taken to indicate statistical significance. All analyses were performed using the SigmaStat Statistical Analysis System software package (Jandel Scientific, San Rafael, CA).
RESULTS
Inhibition of Neurotoxic Esterase Activity Single doses of PSP (5 mg/kg, sc) caused ú97% inhibition of cervical cord NTE 4 hr after treatment in all age groups (Table 1). No additional significant reduction was seen with multiple OP exposures. Extensive NTE inhibition (ú95%) was also noted in birds treated with a single dose of PMSF (90 mg/kg, sc). The different treatment schedules caused similar reductions of enzyme activity across the different age groups. Clinical Signs of OPIDN No clinical signs of OPIDN were observed in any of the 2-week-old birds regardless of the number of PSP treatments or PMSF posttreatment (data not shown). Five-week-old birds (Figs. 1A–1D) were also resistant to OPIDN after receiving only PSP, but PMSF posttreatment was associated with moderate to severe clinical neurotoxicity in all three PSP-treated groups. Single doses of PSP caused minor signs
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FIG. 1. Development of clinical signs of neuropathy in 5-week-old chickens. (A) Ataxia in control birds and birds treated with PMSF (90 mg/kg, sc) only. (B) Compares a single dose of PSP (5 mg/kg, sc) to a single dose of PSP followed 4 hr later by PMSF. (C and D) Compare two or three doses of PSP only to two or three doses of PSP followed by PMSF. Data are shown as median ataxia scores { interquartile ranges. Asterisks indicate significant effects of PMSF posttreatment (i.e., potentiation).
of clinical dysfunction in 8-week-old birds (Figs. 2A–2D) with multiple treatments causing more severe effects than a single dose. PMSF exacerbated delayed neurotoxicity in all 8-week-old birds receiving one, two, or three doses of PSP.
spinocerebellar tracts of the cervical spinal cord. In both tracts the silver-impregnated degeneration appeared as fragmented, irregularly arranged, black silver-impregnated fibers on a light brown or yellow background. The density of the degeneration was dependent on the particular treatment to which the chick was exposed (Fig. 3). No axonal degeneration was found in the cervical cords from any control birds (n Å 4–5 birds/age group; data not shown). Cords from 2-
Morphology Degeneration induced by exposure to PSP appeared consistently and selectively in the gracile fasciculus and dorsal
TABLE 1 Inhibition of Neurotoxic Esterase Activity in Cervical Cords of 2-, 5-, and 8-Week-Old Chickens Treated with Single Doses of PMSF (90 mg/kg, sc) or Single or Repeated Doses of PSP (5 mg/kg, sc) 2-week-old birds
Treatment
n
nmol/min/mg protein
Control PMSF PSP 1 1 PSP 1 2 PSP 1 3
4 4 4 4 3
5.00 0.13 0.14 0.04 0.10
{ { { { {
0.74 0.07 0.02 0.05 0.14
5-week-old birds
% control
n
nmol/min/mg protein
{ { { { {
6 6 6 5 5
9.14 0.30 0.15 00.07 00.15
100.00 2.70 2.88 0.84 1.99
14.87 1.35* 0.42* 0.95* 2.73*
{ { { { {
0.45 0.04 0.05 0.06 0.11
8-week-old birds
% control 100.00 3.29 1.66 00.79 01.68
{ { { { {
4.87 0.47* 0.52* 0.62* 1.26*
n
nmol/min/mg protein
6 4 6 6 6
9.86 0.34 0.11 0.18 0.19
{ { { { {
Note. Data are expressed as means { SE. * Statistically significant at p õ 0.05 by one-way analysis of variance and Student–Newman–Keuls multiple comparison.
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0.38 0.10 0.02 0.04 0.05
% control 100.00 3.48 1.24 1.83 1.90
{ { { { {
3.85 1.04* 0.21* 0.42* 0.49*
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NTE AND POTENTIATION OF OPIDN
FIG. 2. Development of clinical signs of neuropathy in 8-week-old chickens. (A) Ataxia in control birds and birds treated with PMSF (90 mg/kg, sc) only. (B) Compares a single dose of PSP (5 mg/kg, sc) to a single dose of PSP followed 4 hr later by PMSF. (C and D) Compare two or three doses of PSP only to two or three doses of PSP followed by PMSF. Data are shown as median ataxia scores { interquartile ranges. Asterisks indicate significant effects of PMSF posttreatment. Pound signs indicate significant effects of repeated doses of PSP compared to a single dose.
week-old birds had only small amounts of axonal degeneration even following multiple PSP treatments; PMSF posttreatment did not increase degeneration in this age group (Fig. 4A). In the gracile fasciculi from 5-week-old birds, moderate amounts of axonal degeneration were present after single or repeated PSP exposures (Fig. 4B). More extreme (moderate to heavy) degeneration was found in the gracile fasciculi of birds that received both PSP and PMSF. In the spinocerebellar tracts of 5-week-old birds, degeneration was light to moderate in PSP-only treated birds but moderate to heavy in PSP/PMSF-treated birds. Between the two regions, the gracile fasciculus was more severely affected than the spinocerebellar tracts by PSP-only exposures. PMSF posttreatment increased the severity of damage in both regions, with somewhat greater changes (relative to PSP-only) noted in the spinocerebellar tracts. In the gracile fasciculi of 8-week-old birds, degeneration scores ranged from moderate to heavy depending on the number of PSP exposures, with little difference between the PSP-only groups and their respective PMSF posttreated groups (Fig. 4C). In contrast, the spinocerebellar tracts from 8-week-old birds showed light to moderate degeneration in response to PSP only but moderate to heavy degeneration after PSP followed by PMSF.
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DISCUSSION
Inhibition of NTE is the putative first step in initiation of OPIDN (for reviews see Abou-Donia and Lapadula, 1990; Johnson, 1990; Lotti, 1992; Richardson, 1992). In the present studies, single doses of phenyl saligenin phosphate inhibited NTE activity in cervical spinal cord by at least 97%. Subsequent doses of PSP did not significantly increase enzyme inhibition. In contrast to a report by Moretto and co-workers (1991), we found that spinal cord NTE activity in control birds increased with age (5.00 { 0.74, 9.14 { 0.45, and 9.86 { 0.38 nmol/min/mg protein for 2-, 5-, and 8-week-old birds, respectively). Differences in tissue (cervical vs lumbosacral regions) or assay procedures could account for these contrasting results. Two-week-old birds were completely resistant to clinical signs of OPIDN following either single or repeated doses of PSP and exhibited only slight degrees of axonal degeneration, even after PMSF posttreatment. Five-week-old birds were relatively resistant to clinical signs of OPIDN after one, two, or three doses of PSP but developed extensive deficits in locomotor function with PMSF posttreatment in all cases. In general, the degree of axonal degeneration correlated with clinical dysfunction. Similar to previous studies
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using diisopropyl phosphorofluoridate (DFP) as the neuropathic agent (Pope et al., 1993), 8-week-old birds exhibited minimal degrees of clinical dysfunction following a single neuropathic OP exposure. Multiple PSP exposures induced more extensive clinical deficits, however. In addition, PMSF posttreatment amplified the delayed neurotoxicity in 8-weekold chickens initiated by one, two, or three doses of PSP. In 5-week-old chickens, repeated PSP treatments had little effect on the development of clinical signs of neurotoxicity. These findings agree with a previous study (Pope et al., 1993) reporting minimal effects of repeated DFP exposures. In contrast, relatively similar ataxia scores were noted in 8week-old birds treated with either (a) a single dose of PSP followed by PMSF or (b) two doses of PSP. These findings suggest that a subsequent exposure to PSP may be capable of potentiating the neurotoxic effects of a prior dose, but only in more mature animals. Collectively, these studies suggest that some neuropathic OPs may have multiple target sites of relevance to OPIDN and may be capable of both initiation and potentiation of delayed neurotoxicity. The resistance to clinical dysfunction following either repeated PSP or PMSF posttreatment in 2-week-old chickens, the resistance to potentiation by repeated OP exposures but sensitivity to potentiation of both clinical and morphological indicators of OPIDN by PMSF posttreatment in 5-week-old chickens, and finally, the greater sensitivity to both repeated OP exposures and PMSF posttreatment in 8-week-old chickens suggest that an age-related gradient exists in the degree of sensitivity to both initiators and potentiators of delayed neurotoxicity. The results of this study indicate that both the gracile fasciculus and the dorsal spinocerebellar tracts undergo distal axonal degeneration following exposure to PSP. These data are consistent with previous reports indicating that both of these tracts are susceptible to a variety of organophosphorus compounds (Tanaka and Bursian, 1989; Tanaka et al., 1990; Carboni et al., 1992; Dyer et al., 1992). Although both dorsal spinocerebellar and ventral spinocerebellar tracts have been reported in birds (Whitlock, 1952; Karten and Hodos, 1967), the present results indicate that the larger dorsal tract undergoes more severe degeneration after exposure to PSP and PSP/PMSF, a finding similar to that reported in chickens after exposure to tri-ortho-tolyl phosphate (Tanaka and Bursian, 1989) and diisopropylphosphorofluoridate (Tanaka et al., 1990). Previous studies have hypothesized that degeneration of the dorsal spinocerebellar tract, which has been shown in chickens to terminate as mossy fiber afferents in the cerebellar cortex (Okado et al., 1987), may be closely linked to the onset of ataxia (Pope et al., 1993; Funk et al., 1994). The morphological data in the present FIG. 3. Photomicrographs illustrating the different degeneration scores (0–3) noted in the dorsal spinocerebellar tract following administration of PSP and/or PMSF in 8-week-old chickens. (A) The absence of degeneration (degeneration score Å 0) following an injection of PMSF only. (B) The light punctate degeneration (degeneration score Å 1) present after a single injec-
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tion of PSP. (C and D) Illustrate moderate (degeneration score Å 2) to heavy (degeneration score Å 3) degeneration present after injections of both PSP and PMSF. Arrowheads indicate degenerating axons. Scale bar, 50 mm.
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exist. For example, cords from 2-week-old birds had slight amounts of degeneration but the birds were completely resistant to clinical dysfunction. These results agree with a previous study (Funk et al., 1994) following repeated DFP exposures in 2-week-old chickens. Comparison of similarly treated groups of 5- and 8-week-old birds (Figs. 4B and 4C) indicated very similar degrees of axonal damage but the older birds exhibited more severe clinical signs of delayed neurotoxicity. These observations may indicate that younger animals are not just resistant to the initial neuropathic insult but, because of the developing state of their nervous system, are also more successful at compensating for the damage. Although the putative target for initiation of OPIDN is neurotoxic esterase, Pope and Padilla (1990) proposed that potentiation of OPIDN by nonneuropathic NTE inhibitors did not involve NTE. The present study supports this hypothesis by demonstrating that PMSF can amplify clinical and morphological indicators of OPIDN in 5-week-old chickens when given after one, two, or three injections of PSP; dosing regimens which cause from 97 to 100% inhibition of NTE. In 8-week-old birds, PMSF posttreatment also exacerbated clinical dysfunction following similar dosing regimens. In addition, a second dose of PSP markedly increased the delayed neurotoxicity of an initial dose of the OP. More recently, Moretto and co-workers (1994) demonstrated potentiation of OPIDN by O-(2-chloro-2,3,3-trifluorocyclobutyl) Oethyl S-propyl ester (KBR-2822) at doses which did not significantly affect NTE activity. Collectively, these studies support the hypothesis that another target molecule is involved in potentiation of OPIDN. In conclusion, we have demonstrated that PMSF can potentiate the delayed neurotoxic effects of PSP in the presence of less than 3% residual NTE activity. The results further support the hypothesis that potentiation of OPIDN involves a target molecule other than NTE. FIG. 4. Axonal degeneration in the dorsal columns (shaded bars) and spinocerebellar tracts (striped bars) of cervical cords from 2-, 5-, and 8-weekold chickens treated with PSP (5 mg/kg, sc) with or without PMSF (90 mg/ kg, sc). Tissues were collected 16 days after treatment and examined for degeneration using a modification of the Fink–Heimer silver staining technique. Morphological effects in 2-week-old chickens (A) were measured in birds receiving either three doses of PSP or three doses of PSP followed by PMSF 4 hr after the final treatment. Axonal degeneration was examined in 5- (B) and 8- (C) week-old birds following one, two, or three doses of PSP with or without PMSF posttreatment. No axonal degeneration was noted in any samples from any age group treated with vehicles or PMSF alone.
study add to the results from these previous investigations by showing that although PMSF posttreatment increases degeneration in both regions of the cervical cord, it appears to produce greater changes (relative to OP exposure only) in the dorsal spinocerebellar tract. While the morphological data in the present study generally support the clinical findings, some interesting contrasts
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ACKNOWLEDGMENTS The authors appreciate the technical assistance of Mr. Thuc Pham and Dr. Tamal Chakraborti. This research was partially supported through cooperative agreement CR820229-01 between the United States Environmental Protection Agency and Northeast Louisiana University (C.N.P.).
REFERENCES Abou-Donia, M. B., and Lapadula, D. M. (1990). Mechanisms of organophosphorus ester-induced delayed neurotoxicity: Type I and Type II. Annu. Rev. Pharmacol. Toxicol. 30, 405–440. Bondy, H. F., Field, E. J., Worden, A. N., and Hughes, J. P. W. (1960). A study on the acute toxicity of the tri-aryl phosphates used as plasticizers. Br. J. Ind. Med. 17, 190–200. Carboni, D., Ehrich, M., Dyer, K., and Jortner, B. S. (1992). Comparative evolution of mipafox-induced delayed neuropathy in rats and hens. Neurotoxicology 13, 723–734. Caroldi, S., Lotti, M., and Masutti, A. (1984). Intraarterial injection of diisopropylfluorophosphate or phenylmethane sulfonyl fluoride produces
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unilateral neuropathy or protection, respectively, in hens. Biochem. Pharmacol. 33, 3213–3217. Dyer, K., Jortner, B. S., Shell, L. G., and Ehrich, M. (1992). Comparative dose–response studies of organophosphorus ester-induced delayed neuropathy in rats and hens administered mipafox. Neurotoxicology 13, 745– 756. Funk, K. A., Henderson, J. D., Liu, C. H., Higgins, R. J., and Wilson, B. W. (1994). Neuropathology of organophosphate-induced delayed neuropathy (OPIDN) in young chicks. Arch. Toxicol. 68, 308–316. Johnson, M. K. (1970). Organophosphorus and other inhibitors of brain ‘‘neurotoxic esterase’’ and the development of delayed neurotoxicity in hens. Biochem. J. 120, 523–531. Johnson, M. K., and Barnes, J. M. (1970). Age and the sensitivity of chicks to the delayed neurotoxic effects of some organophosphorus compounds. Biochem. Pharmacol. 19, 3045–3047. Johnson, M. K. (1974). The primary biochemical lesion leading to the delayed neurotoxic effects of some organophosphorus esters. J. Neurochem. 23, 785–789. Johnson, M. K. (1977). Improved assay of neurotoxic esterase for screening organophosphates for delayed neurotoxicity potential. Arch. Toxicol. 37, 113–115. Johnson, M. K. (1982). The target for initiation of delayed neurotoxicity by organophosphorus esters: Biochemical studies and toxicological applications. In Reviews in Biochemical Toxicology (E. Hodgson, J. R. Bend, and R. M. Philpot, Eds.), Vol. 4, pp. 141–212. Elsevier, New York. Johnson, M. K. (1990). Organophosphates and delayed neuropathy—Is NTE alive and well? Toxicol. Appl. Pharmacol. 102, 385–399. Jortner, B. S., and Ehrich, M. (1987). Neuropathological effects of phenyl saligenin phosphate in chickens. Neurotoxicology 8, 303–314. Karten, H. J., and Hodos, W. (1967). A Stereotaxic Atlas of the Brain of the Pigeon (Columbia livia), 193 pp. Johns Hopkins Press, Baltimore. Lotti, M. (1992). The pathogenesis of organophosphate polyneuropathy. Crit. Rev. Toxicol. 21, 465–487. Lotti, M., Caroldi, S., Capodicasa, E., and Moretto, A. (1991). Promotion of organophosphate-induced delayed polyneuropathy by phenylmethanesulfonyl fluoride. Toxicol. Appl. Pharmacol. 108, 234–241. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Moretto, A., Capodicasa, E., Peraica, M., and Lotti, M. (1991). Age sensitivity to organophosphate-induced delayed polyneuropathy. Biochem. Pharmacol. 41, 1497–1504. Moretto, A., Bertolazzi, M., and Lotti, M. (1994). The phosphorothioic acid O-(2-chloro-2,3,3-trifluorocyclobutyl) O-ethyl S-propyl ester exacerbates organophosphate polyneuropathy without inhibition of neuropathy target esterase. Toxicol. Appl. Pharmacol. 129, 133–137. Okado, N., Ito, R., and Homma, S. (1987). The terminal distribution pattern of spinocerebellar fibers: An anterograde labelling study in the posthatching chick. Anat. Embryol. 176, 175–182.
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Peraica, M., Capodicasa, E., Moretto, A., and Lotti, M. (1993). Organophosphate polyneuropathy in chicks. Biochem. Pharmacol. 45, 131–135. Pope, C. N., and Padilla, S. S. (1989). Chromatographic characterization of neurotoxic esterase. Biochem. Pharmacol. 38, 181–188. Pope, C. N., and Padilla, S. (1990). Potentiation of organophosphorus-induced delayed neurotoxicity by phenylmethylsulfonyl fluoride. J. Toxicol. Environ. Health 31, 261–273. Pope, C. N., Chapman, M. L., Tanaka, D., Jr., and Padilla, S. (1992). Phenylmethylsulfonyl fluoride alters sensitivity to organophosphorus-induced delayed neurotoxicity in developing animals. Neurotoxicology 13, 355– 364. Pope, C. N., Tanaka, D., Jr., and Padilla, S. (1993). The role of neurotoxic esterase (NTE) in the prevention and potentiation of organophosphorusinduced delayed neurotoxicity (OPIDN). Chem.-Biol. Interact. 87, 395– 406. Richardson, R. J. (1992). Interactions of organophosphorus compounds with neurotoxic esterase. In Organophosphates: Chemistry, Fate, and Effects (J. E. Chambers and P. E. Levi, Eds.), pp. 299–323. Academic Press, San Diego. Roberts, N. L., Fairley, C., and Phillips, C. (1983). Screening, acute delayed and subchronic neurotoxicity studies in the hen: Measurements and evaluations of clinical signs following administration of TOCP. Neurotoxicology 4, 263–270. Tanaka, D., Jr. (1976). Thalamic projections of the dorsomedial prefrontal cortex in the Rhesus monkey (Macaca mulatta). Brain Res. 110, 21–38. Tanaka, D., Jr., and Bursian, S. J. (1989). Degeneration patterns in the chicken central nervous system induced by ingestion of the organophosphorus delayed neurotoxin tri-ortho-tolyl phosphate: A silver impregnation study. Brain Res. 484, 240–256. Tanaka, D., Jr., Bursian, S. J., and Lehning, E. (1990). Selective axonal and terminal degeneration in the chicken brainstem and cerebellum following exposure to bis(1-methylethyl) phosphorofluoridate (DFP). Brain Res. 519, 200–208. Tanaka, D., Jr., Bursian, S. J., and Lehning, E. J. (1992a). Neuropathological effects of triphenyl phosphite in the central nervous system of the hen (Gallus domesticus). Fundam. Appl. Toxicol. 18, 72–78. Tanaka, D., Jr., Bursian, S. J., and Lehning, E. J. (1992b). Silver impregnation of organophosphorus-induced delayed neuropathy in the central nervous system. In The Vulnerable Brain and Environmental Risks. Vol. 2. Toxins in Food (R. Isaacson and K. Jensen, Eds.), pp. 215–234. Plenum, New York. Varghese, R. G., Bursian, S. J., Tobias, C., and Tanaka, D., Jr. (1995). Organophosphorus-induced delayed neurotoxicity: A comparative study of the effects of tri-ortho-tolyl phosphate and triphenyl phosphite on the central nervous system of the Japanese quail. Neurotoxicology 16, 45– 54. Veronesi, B., and Padilla, S. (1985). Phenylmethylsulfonyl fluoride protects rats from mipafox-induced delayed neuropathy. Toxicol. Appl. Pharmacol. 81, 258–264. Whitlock, D. G. (1952). A neurohistological and neurophysiological study of afferent fiber tracts and receptive areas of the avian cerebellum. J. Comp. Neurol. 97, 567–635.
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