http://jhc.sagepub.com/ Journal of Histochemistry & Cytochemistry http://jhc.sagepub.com/content/37/5/589 The online version of this article can be found at: DOI: 10.1177/37.5.2703698 1989 37: 589 J Histochem Cytochem G B Koelle, N S Thampi, M S Han and E J Olajos Histochemical demonstration of neurotoxic esterase. Published by: http://www.sagepublications.com On behalf of: Official Journal of The Histochemical Society can be found at: Journal of Histochemistry & Cytochemistry Additional services and information for http://jhc.sagepub.com/cgi/alerts Email Alerts: http://jhc.sagepub.com/subscriptions Subscriptions: http://www.sagepub.com/journalsReprints.nav Reprints: http://www.sagepub.com/journalsPermissions.nav Permissions: What is This? - May 1, 1989 Version of Record >> by guest on December 20, 2011 jhc.sagepub.com Downloaded from
9
Embed
His to Chemical Demonstration of Neurotoxic Esterase.
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
http://jhc.sagepub.com/Journal of Histochemistry & Cytochemistry
http://jhc.sagepub.com/content/37/5/589The online version of this article can be found at:
DOI: 10.1177/37.5.2703698
1989 37: 589J Histochem CytochemG B Koelle, N S Thampi, M S Han and E J Olajos
Histochemical demonstration of neurotoxic esterase.
Published by:
http://www.sagepublications.com
On behalf of:
Official Journal of The Histochemical Society
can be found at:Journal of Histochemistry & CytochemistryAdditional services and information for
Histochemical Demonstration of Neurotoxic Esterase’
GEORGE B. KOELLE,2 NAGENDRAN S. THAMPI, MATTHEW S. HAN,
and EUGENE J. OLAJOS3
Department ofPharmacology, Medical School, University ofPennsylvania, Philadelphia, Pennsylvania 19104-6084.
Received for publication August 17, 1988 and in revised form November 14, 1988; accepted November 22, 1988 (8A1462).
We developed a histochemical method for localizing neuro-toxic esterase (NTh), defined as the phenylvalerate (PV)-hydrolyzing esterase that is resistant to 40 �tM paraoxon (A)but inactivated by paraoxon plus 50 �iM impafox (B). NThis considered to be the target enzyme in the production
of organophosphorus ester-induced delayed neurotoxicity(OPIDN). Cryostat sections were incubated in a medium con-taming a-naphthyl valerate and 6-benzamido-4-methoxy-m-toluidine diazonium chloride (fast violet B) after treatmentwith the above-mentioned inhibitors, leading to formationof an aqueous insoluble precipitate at sites of enzymatic ac-tivity. NTh activity was estimated as staining detectable in
is a syndrome produced by certain compounds of that category.
It is characterized by sequential development of axonal degenera-
tion, demyelination, and flaccid paralysis after a latent period of
10-14 days, and is completely unrelated to the anticholinesterase
(anti-ChE) action of such compounds. Several extensive outbreaks
of human OPIDN have been reported, usually resulting from the
contamination of beverages or cooking oil with triorthocresyl phos-
phate. The subject has been reviewed thoroughly by Abou-Donia
(1981) and Zech and Chemnitius (1987).
Although its biochemical basis is still unproven, one ofthe most
generally considered proposals at present is that OPIDN is caused
by alkylphosphorylation and subsequent “aging” (partial dealky-
lation) of neurotoxic esterase (NTh) Uohnson, 1969, 1977). This
enzyme has been defined empirically by Johnson (1977) as the
phenylvalerate (PV)-hydrolyzing esterase that is resistant to 40 p.tM
paraoxon (0,0-diethyl-0-p-nitrophenyl phosphate; E 600) but is in-
activated by paraoxon plus 50 �sM mipafox (N,N-diisopropyl
fluorophosphorodiamide). An additional fraction of PV-hydrolyzing
1 Supported by Contract DAAA15-87-K-0002, Department of the
Army.2 To whom correspondence should be addressed.
3 Present address: Chemical Research, Development and Engineering
Center, US Army, Aberdeen Proving Ground, MD 21010-5423.
A but not in B. In the central nervous system (CNS) ofchicken, NTh appeared to be present primarily in the so-mata of most neurons, but at sites indistinguishable fromthose of the other inhibitor.resistant and -sensitive a-naph.thyl valerate-hydrolyzing esterases. It could not be distin-guished in the CNS of cat, probably because it constitutes
less than 3% ofthe total PV.hydrolyzing activity in the CNSofthat species. (JHistochem Cytochem 37:589-596, 1989)
KEY WORDS: a-Naphthyl valerate; Cat; Central nervous system;
Chicken; Fast violet B; Neurotoxic esterase (NTh); Organophos-
addition of phenyl valerate was omitted, were included. All determina-
tions were done in duplicate. The mixtures were incubated for exactly 20
mm at 37C with shaking. To each tube was then added 2.5 ml of phenyl
valerate reagent (9 mg phenyl valerate dissolved in 1.0 ml dimethylforma-
mide plus 30 ml 0.03% Triton X-100). After incubation at 37’C for 30 mm,
2.5 ml ofcold 0.3 M pcrchloric acid was added to each tube and the mix-
tures were cooled in an icc-water bath. After centrifugation at 3500 rpmfor 10 mm in the cold, 4 ml clear supernatants were added to tubes con-
taming 2 ml 4-aminoantipyrinc solution (0.05% in 0.5 M Tris buffer, pH
9.0); after thorough mixing, 1.0 ml 0.4% K3Fc(CN)�j was added, result-
ing in immediate development of a plum-red color. Absorbance was read
in a Beckman spectrophotometer at 510 nm. A standard curve was obtained
by plotting the absorbance at 510 nm against various concentrations of phenolafter reaction with aminoantipyrinc and K�Fe(CN)� under conditions iden-tical with those described above.
Histochemical. Of the several procedures that have been used for lo-calization ofcstcrases(Deimling and Backing, 1976; Pcarsc, 1973), the highlysensitive, simultaneous coupling a.zo dye methods appeared to be the most
promising. This approach was introduced by Menten ct al. (1944) for local-
ization of alkaline phosphatase, and was first applied to carboxylic acidcsterases by Nachlas and Seligman (1949). Since then many improvements
and modifications have been proposed. a-Naphthyl esters are generally em-
ployed as substrates, because a-naphthol rapidly forms insoluble precipi-tates with the diazonium salts of several amines. The disadvantage of this
method in the present case is that hen brain has been shown to hydrolyze
a-naphthyl valcratc (a-NV) at only one sixth the velocity for PV, and theparaoxon-rcsistant, mipafox-sensitive portion constitutes only 5 % of the
total in contrast to 50% reported for PV Uohnson, 1975a).
Twenty-one diazonium salts, all purchased from Sigma. were tested ascoupling agents in histochemical experiments with chicken spinal cord orbrain similar to those described below. The list, with catalog (1987) num-
bets, included: fist black K (7253), fast blue BB (3378), f�.st blue RR (0500),fast bordeaux (1005), fast corinth V (6383), fast dark blue R (0750), fast
garnet GBC (6504), fast orange GR (3137), fast red AL (5002), fast red B
(3262), fast red 3GL (0380), fast red ITR (1375), fast red KL (8379), fast
red PDC (6876), fast red RC (2256), fast red RI. (1630), fast red TR (6760),
fast red violet LB (3381), fast scarlet GG (9379), fast scarlet R (1880), and
fast violet B (1631). The most satisfactory appeared to be the last mentioned,which is chemically 6-benzamido-4-mcthoxy-m-toluidine diazonium chlo-
ride (fast violet B salt; P/B). This is consistent with the rating given forthis compound among 23 tested by Pcarsc (1973), as a coupling agent for
localization ofacid phosphatases. Other variables tested included fixatives
and conditions of fixation; concentrations of reagents; pH; time and tern-peraturc ofincubation; and mounting media. After 29 preliminary experi-
ments with serial sections of chicken spinal cord and seven with chicken
brain, the following procedure was adopted.The brain and spinal cord ofRhodc Island Red hens weighing 1.5-2.5
kg (decapitated) and cats (sacrificed by means of sodium pentobarbital,
50 mg/kg, iv, followed by thoracotomy) were removed and sectioned im-mediately or after a few days storage at - 70’C. Sections were cut at 20
�am in a cryostat and placed on slides coated with 2% bovine serum albu-
mm the previous day. After drying for approximately 1 hr, slides were im-
messed in unbuffered 1% formaldehydc/0.9% NaCI at 5C for 10 mm and
rinsed twice for 10 mm in cold 0.9% NaCI. (This distinctly improved stain-ing in contrast to unfixed sections; longer fixation, higher concentrations
of formaldehyde, or any concentration of glutaraldehyde caused markedinhibition of enzyme activity.) With this limited degree of fixation and
the prolonged times ofincubation required, structural preservation was neces-sarily compromised. After drying for 1 hr at room temperature, slides were
prc-incubatcd for 20 mm at 37C in the following solutions, containing
0.9% NaCI: A, control; B, 40 isM paraoxon; C, 40 �iM paraoxon plus 50
�tM mipafox; D, 40 �tM paraoxon plus 50 piM mipafox plus 50 laM DFP
(Paraoxon and DFP were prepared as stock solutions in anhydrous acetone
and propylene glycol, respectively, and held in desiccators in the refrigera-tor for a maximum of 4 weeks; mipafox was prepared as an aqueous solu-
tion extemporaneously.) After two brief rinses in 0.9% NaCl, slides were
dried for 1 hr in the hood, then immersed in the following incubation so-
lution at room temperature for 1-4 hr: P/B, 40 mg; 50 mM Tris-0.2 mM
EDTA buffer, pH 8.0, 32 ml; a-NV (0.1 ml in 20 ml dimethylformamide),
8 ml. One minute after addition ofa-NV the solution was filtered (What.
man No. 1) into Coplin jars and the slides were added. Slides were placed
in fresh incubation solution hourly. After removal, they were rinsed briefly
in distilled water and allowed to dry overnight. They were then mounted
directly in glycerin jelly or Permount and examined. (The precipitate is
soluble in alcohol.)
This final procedure was employed in nine experiments with serial 5cc-
tions ofchickcn brain, and two each with chicken spinal cord, sciatic nerve,
ileum, kidney, and liver. Serial sections of cat CNS were studied in five
experiments. In each experiment a total of 16 slides, containing three to
six sections each, were stained.
By definition, NTh activity was estimated as staining detectable after
pre-incubation in B but not in C; A shows all a-NV-hydrolyzing esterases;
D shows a-NV-hydrolyzing esterases resistant to all three inhibitors, including
DFP
Results
Quantitative
In the CNS ofthe chicken, the proportion ofPV-hydrolyzing activ-ity designated as NTh was found to constitute approximately 6%
of the total (Table 1). As noted previously with respect to the hu-
man brain (Lotti and)ohnson, 1980), there was no significant differ-
ence between various regions. The values obtained here are approx-
imately half those reported by Novak and Padilla (1986) and
considerably lower than those found byJohnson (1975b) and Reveley
et al. (1986); the reasons for these discrepancies are not apparent.
The sciatic nerve was found to contain less than half the NTh ac-
tivity of the CNS. In the non-neural parenchymatous tissues ana-
lyzed (ileum, kidney, liver), the PV-hydrolyzing activity was two
to three times that of the brain but only traces ofNTE activity were
found.
Values for NTh in the CNS ofthe cat ranged between only 1-3%
of total PV-hydrolyzing activity.
Histochemical
Figure 1 illustrates at low magnification the sequential effects of
the inhibitors employed on the total pattern of staining in cross-
sections ofchicken cervical spinal cord. In the absence of inhibitors,
incubation for 4 hr resulted in intense staining for a-NV esterase
in what appeared to be the penikarya of essentially all neurons in
the central gray matter (Figure 1A). The initial portions of their
axons could sometimes be followed for short distances into the white
matter adjacent to the anterior horns; however, staining in this re-
gion was not marked. In addition, marked a-NV esterase staining
occurred in the pia-arachnoid membranes, where they were ad-
herent, and in the Virchow-Robin spaces surrounding the arteries,
as both longitudinally and frequently transversely cut patterns
throughout the white matter. Preliminary treatment with paraoxon
(Figure 1B) resulted in marked reduction of intensity at the above
by guest on December 20, 2011jhc.sagepub.comDownloaded from
Figure 1. Transverse 2O-�tm sections of cervical spinal cord of chicken stained by incubation for 4 hr at 20#{176}Cin a solution containing a-NV plus RIB. Prior treatmentfor 20 minutes at 37#{176}Cwith the following inhibitors, from lower left, clockwise: (A) none; (B) 40 RM paraoxon; (C) paraoxon plus 50 �tM mipafox; (D) paraoxonplus mipafox plus 50 sM DFR NTE is identified as staining detectable in B minusthat in C. Although staining is progressively lighter in A through D, no qualitativechange can be detected in its distribution. Original magnification x 27. Bar = 500 �tm.
592 KOELLE, THAMPI, HAN, OLAJOS
by guest on December 20, 2011jhc.sagepub.comDownloaded from
Figure 2. Higher magnification of identical regions of the anterior horn as shown in sections in Figure lA-iD. Original magnification x 437. Bar = 20 �sm.
.�.,
tI
by guest on December 20, 2011jhc.sagepub.comDownloaded from
Chemnitius 3M, Zech R (1983a): Inhibition of brain carboxylcstcrases by Lotti Mjohnson MK (1978): Neurotoxicity oforganophosphorus pesticides:neurotoxic and non-neurotoxic organophosphorus compounds. Mol Phar- predictions can be based on in vitro studies with hen and human enzymes.
macol 23:717 Arch Toxicol 41:215
ChemnitiusJM, Zech R (1983b): Carboxylesterases in primate brain: char- Menten ML,JungeJ, Green MH (1944): A coupling histochemical azo dye
acterization of multiple forms. mt J Biochem 15:1019 test for alkaline phosphatase in the kidney. J Biol Chcm 153:471
Deimling OV, Bocking A (1976): Esterases in histochemistry and ultra- Nachlas MM, Seligman AM (1949): The histochemical demonstration ofhistochemistry. Histochem J 8:215 esterase. j NatI Cancer Inst 9:415
Johnson MK (1980): Neurotoxicity: mechanisms explored and exploited.Novak R, Padilla S (1986): An in vitro comparison ofrat and chicken brainNature 287:105neurotoxic esterase. Fundam Appl Toxicol 6:464
J ohnson MK (1977): Improved assay of neurotoxic esterase for screeningorganophosphatcs for delayed neurotoxicity potential. Arch Toxicol 37:113 Pearse AGE (1973): Histochemistry. Theoretical and applied. Vol 2. 3rd
ed. Edinburgh, Churchill Livingstonc, 761Johnson MK (1975a): Structure-activity relationships for substrates and in-hibitors of hen brain neurotoxic cstcrasc. Biochem Pharmacol 24:797 RcveleyJW, Sabourin TD, Moore MT. Goss LB(1986): Distribution of ncu-
Johnson MK (1975b): The delayed neuropathy caused by some organo- rotoxic esterase activicy in the brain of control and diisopropyl phos-
phosphorus esters: mechanism and challenge. CRC Crit Rev Toxicol 3:289 phorofluoridate-treated hens: in vivo and in v#{252}mexposure. Toxicol Len 31:45
Johnson MK (1975c): Organophosphorus esters causing delayed neurotoxic Thomas E (1977): Histochemic der Enzyme in peripheren Nervensystem.
effects: mechanism ofaction and structure/activity relationships. Arch Tox- Stuttgart, Gustav Fischer Verlag, 47, 62icol 34:259
Yoshikawa T(1968a): Atlas ofthc brains ofdomcstic animals. Tokyo, Univer-Johnson MK(1969): The delayed neurotoxic effrct ofsome organophosphorus sity of Tokyo Press, F25
compounds - identification of the phosphorylation site as an esterase. Bio-chem J 114:711 Yoshikawa T(1968b): Atlas ofthc brains ofdomestic animals. Tokyo, Univer-
Lotti M, Becker CE, Aminoff MJ (1984): Organophosphate polyncuropa- sity of Tokyo Press, F17, F18