X-Ray Studies on Phospholipid Bilayers. XII. Interactions of Pentachlorophenol with Myelin M . Suwalsky, F. Villena3, G. M o n to y ab, C. G a rrid o , I. Sá nchez, and F. N eira Department of Chemistry, aDepartment o f Histology and bDepartment of Pharmacology, University of Concepcion, Casilla 3-C, Concepcion, Chile Z. Naturforsch. 47c, 601-607 (1992); received February 6, 1992 X-Ray Diffraction, Phospholipid Bilayer, Pentachlorophenol, Myelin Pentachlorophenol (PCP) is a widely used pesticide, particularly for the preservation of wood. Given its high toxicity and resistance to degradation it has become a dangerous envi ronmental pollulant. Due to its high lipophilicity, PCP is able to partition into the lipid bilayer of cell membranes disrupting several vital functions. The present research was concerned with the effects that the chronic administration o f PCP could produce in vivo to the sciatic nerve of rats. X-ray diffraction patterns obtained from freshly dissected and dried sciatic nerves of PCP treated rats did not show significant differences in their reflections with respect to those pres ent in the patterns from untreated animals. However, morphological studies performed by optical and electron microscopy showed degenerative changes in about 10% of the A and B type of nerve fibers.
Introduction P entach lo ro p h en o l (PCP) an d its sodium salt are widely used fungicides, p articularly for the preservation o f w ood. G iven their high toxicity an d resistance to d egradation they have becom e d angerou s environm ental pollulants [1 -4 ], P C P is readily abso rb ed th ro ug h the skin and the diges tive a n d respiratory systems. A s it is strongly lipo philic it is able to p artitio n in to the lipid bilayer o f cell m em branes disrupting several functions. T hus, it uncouples the oxidative pho sp h o rilatio n [5], al ters the m icrosom al electron tra n sp o rt system [6 ] an d inhibits the am ino acid tra n sp o rt across cell m em branes [7], As a result o f treatm en t w ith sublethal doses o f PC P, the lipid bilayers o f cell m am m alian m em branes becam e destabilized [8 ]. S tru ctu ral studies on the interaction o f PC P w ith phosph o lip id bilayers have been perform ed in this lab o ra to ry by X -ray diffraction techniques [9]. M ultibilayers built up o f dim yristoylphosphatidylcholine (D M P C ) and dim yristoylphosphatidyleth anolam in e (D M P E ), phospholipids th a t are re spectively present in the outer and inner m o n o lay ers o f cell m em branes, were used. It was observed th a t P C P strongly p erturbed D M P C and D M P E m olecular structures in a hydrophobic as well as in Reprint requests to Dr. M. Suwalsky. Verlag der Zeitschrift für Naturforschung, D-W-7400 Tübingen 0939-5075/92/0700-0601 $ 01.30/0
a hydrophilic m edium . In view o f these results, it was considered o f interest to study the effects th a t the chronic ad m in istratio n o f PC P could produce in vivo to the stru ctu re o f cell m em branes. F o r this purpose, ra ts were allow ed to drink ad libitum aqueous solutions o f P C P for 9 0 -1 2 0 days. This p ap er presents the results o f the X -ray studies per form ed on freshly dissected and air-dried sciatic nerves o f rats treated an d u n treated w ith PCP w ater solutions. O ptical an d electron m icroscopy observations were also m ade on these specimens.
Materials and Methods M ale W istar albino ra ts o f 3 0 0 -3 5 0 gram s o f body w eight were divided into four groups. The first was given PC P (Sigm a, lot 127 F 3439) 1.0 m M for 90 days; the second, 3.0 m M for 120 days; the th ird received 10.0 m M PC P until the rats showed the sym ptom s o f intoxication and the fo u rth was a co n tro l group. E ach anim al was killed by decapi ta tio n , b o th sciatic nerves were rem oved and kept u n d er slight tension in sam ple holders. T he X -ray diagram s were ob tain ed in a W arhus cam era p ro vided w ith a 0.38 m m diam eter pinhole collim ator. O ne o f each p air o f freshly dissected nerves was inm ediately set in the cam era and X -ray diffracted at 18 °C and a b o u t 100% o f relative hum idity. This was atta in ed by bubbling air first th ro u g h two w ater-containing gas-w ashing bottles. The other nerve w as dried in the am bient atm osphere by
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M. Suwalsky et al. ■Interactions o f Pentachlorophenol with Myelin
keeping it for several days at 15 °C an d then X -ray diffracted und er vacuum . Sam ple-to-film distances were either 8 cm o r 29 cm. N i-filtered C u K a ra d iatio n from a Philips PW 1140 g en erato r was used. The relative intensities o f the reflections were m easured from the X -ray films using a JoyceLoebl M K III CS m icrodensitom eter connected to an A cer 915 m icrocom puter. F o r the electron m icroscopy o bservations, the nerves were inm ediately fixed in 2.5% glutaraldehyde in 0.15 m sodium p h o sp h ate buffer for 30 m in an d post fixed w ith 2 % osm ium tetroxide for 1 h. A fter a short rinsing w ith buffer, the specimens were dehydrated in graded eth an o l series, em bed ded in E pon and processed for u ltra thin section using a M T 2 P o rter Blum u ltram icro to m e (D u P o n t Instr., Sorvall). Sections fo r electron m icro scopy were stained w ith a 2 % aq u eo u s solution o f uran y l acetate and lead citrate stain. T hick sec tions for light m icroscopy were stained w ith toluidine blue. T ransm ission electron m icroscopy o b servations were m ade in a Philips 200 EM , while a Leitz O rth o p lan was used for o ptical m icroscopy. Results Fig. 1 and 2 show the high-angle X -ray o riented diagram s o f dry sciatic nerves respectively o b tained from norm al and PC P fed rats. A co m p ari son o f their in terp lan ar spacings, o rien tatio n o f the reflections and their intensities, including those obtain ed from low -angle X -ray diagram s, are pres ented in Table I. As it can be observed, there are no significant differences betw een th e p a tte rn s o f b o th nerves. A b o u t the sam e results were o b tained w hen freshly dissected sciatic nerves were exposed
Fig. 2. High-angle X-ray diagram of a dry sciatic nerve of a rat treated with 10 m M PCP for 120 days. D = 8 cm.
to X -ray diffraction. These patterns, however, show ed fewer reflections and they were m ore dif fuse th a n those obtained from the dry specimens. T able II shows a com parison o f the spacings and intensities o f fresh nerves from PC P u n treated and treated rats. These results, obtained from dry and fresh nerves, were essentially the same independent o f w hether the rats were fed w ith PCP free w ater or 1, 0, 3.0 or 10.0 m M PC P solutions. O bservation m ade by optical and electron m i croscopy show ed, how ever, different results. In fact, the sciatic nerves o f rats fed with 1.0 m M PC P for 90 days and 3.0 mM for 120 days presented de generative changes in a b o u t 10% o f the A and B type o f nerve fibers. As it can be observed in Fig. 3 to 6 , the dam age to the neural com ponents consist ed in various degrees o f u ltrastructural degenera tive changes o f the myelin sheath. These changes vary from fibers w ith incipient and sectorized al teratio n s o f the neuroglial coat (Fig. 4) to fibers presenting a to tal disruption o f the classical lam el lar and periodical structure o f myelin (Fig. 5). In addition, a severe loss o f neurofilam ents, n eu ro tu bules, vesicles and other axoplasm ic com ponents were observed when com pared with norm al myelin fibers (Fig. 6 ). Discussion
Fig. 1. High-angle X-ray diagram of a dry sciatic nerve of a rat without treatment with PCP. D = 8 cm.
X -ray diagram s were obtained from the sciatic nerves o f rats th at dru n k PCP containing w ater w hose concentration ranged between 1 . 0 and 10.0 m M for 90 to 120 days. They were com pared w ith those diagram s obtained from rats th at dru n k
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Table I. Comparison o f observed interplanar spacings (do) and relative intensities (Io rel) o f dehy drated sciatic nerves of rats untreated and treated with PCP1. Dry sciatic nerve of rats untreated with PCP do [Ä] Io rel Orientation 152.3*
76.2* 63.1* 43.7* 33.5 31.6 21.0 16.8 15.9 14.0 12.7 11.4 6.9 6.3 5.8 5.2 5.1 4.9 4.6 4.19
47 88 100 62 5 2 12 3 4 2 1 23 2 5 6 9 4 11 2 34
equatorial equatorial equatorial equatorial + diagonal equatorial equatorial equatorial equatorial equatorial equatorial + meridional equatorial equatorial + meridional equatorial equatorial unoriented meridional meridional meridional meridional meridional
Dry sciatic nerve of rats treated with PCP do [Ä] Io rel Orientation 152 * 76.2* 64.0* 43.7* 32.7 31.2 20.9 16.7 16.0 13.8 12.7 11.4 6.9 6.3 5.8 5.2 5.1 4.9 4.6 4.19
37 84 100 36 7 3 10 6 4 3 4 15 3 6 10 6 3 13 1 81
equatorial equatorial equatorial equatorial + diagonal equatorial equatorial equatorial equatorial equatorial equatorial + meridional equatorial equatorial + meridional equatorial equatorial unoriented meridional meridional meridional meridional meridional
1 The interplanar spacings and intensities o f the reflections were measured in X-ray diagrams ob tained from flat-plate cameras. D = 8 and (*) 29 cm.
Table II. Comparison of observed interplanar spacings (do) and relative intensities (Io rel) of freshly dissected sciatic nerves of rats untreated and treated with PCP1. Sciatic nerve of rat untreated with PCP do [Ä] Io rel Orientation
Sciatic nerve of rat treated with PCP do [Ä] Io rel Orientation
157* 88* 58* 44 33 16 13 4.5
157* 86* 58* 43 32 16 13 4.5
28 66 100 9 2 2 31 11
equatorial equatorial equatorial equatorial equatorial equatorial equatorial unoriented2
15 50 100 3 2 3 37 24
equatorial equatorial equatorial equatorial equatorial equatorial equatorial unoriented2
1 The interplanar spacings and intensities o f the reflections were measured in X-ray diagrams ob tained from flat-plate cameras. D = 8 and (*) 29 cm. 2 Broad and diffuse.
P C P free w ater. Tw o type o f nerves were analyzed. O ne th a t was subjected to X -ray diffraction at 18 °C in a high hum idity atm osphere inm ediately afte r being dissected. T heir p attern s show ed a b o u t h a lf a dozen o f m ostly diffuse and equ ato rially o ri ented reflections, all o f them orders o f a repeat pe rio d o f a b o u t 176 Ä except one o f 157 Ä (T able II). T hey also show ed a b ro a d and unorien ted diffuse ring o f 4.5 Ä. T his reflection, w hich is usually as
sociated to a liquid-like co n fo rm atio n o f the ph o s pholipid h y d ro ca rb o n chains [ 1 0 ], indicated th at they presented the L a phase in the nerve myelin. The o th er type o f specim en consisted o f sciatic nerves th a t were left do dry at 15 °C under slight tension. T heir X -ray diagram s differed from those produced by the fresh sam ples in th a t the latter show ed m ore an d better defined reflections. As it has been rep o rted , the extrem e dehydration o f
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M. Suwalsky et al. • Interactions of Pentachlorophenol with Myelin
Fig. 3. Thick section of a sciatic nerve o f a rat treated with 1.0 mM PCP for 90 days. Three myelinated nerve fibers showing different degrees of alteration are observed (arrows). Optical microscopy; toluidine blue staining, 845 * . (All the micrographs correspond to the same group o f rats.)
Fig. 4. Initial degenerative state of myelinated nerve fiber. Middle myelin layers showing disgregation (ar row). Transmission electron microscopy, 7200 *.
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Fig. 5. Advanced degener ative state o f myelinated nerve fiber (arrow). The regular appearance o f the typical myelin sheath is completely lost. Transmis sion electron microscopy, 7200 x .
Fig. 6. Normal myelinated nerve fiber. Transmission electron microscopy, 20,000 x .
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myelin leads to an irreversible segregation o f its various com p o n en ts [11]. T he diffraction p attern s from such m ixed stru ctu res are difficult to u n scram ble. X -ray p a tte rn s from pure lipid, lipid ex tracts, m ixture o f lipids [ 1 2 ], dry m yelin [ 1 1 ] and the investigation o f the effects o f v arious solvents on the stru ctu re o f myelin [13] have provided the basis for the u n d erstan d in g o f the n a tu re o f the v arious p rotein-lipid m ixtures th a t give rise to the different rep eat periods in the X -ray diffraction diagram s o f the d ry nerves [11]. T hus, the reflec tions present in T able I could be assigned in T a ble III to (a) a d ouble bilayer w ith p ro tein in the bilayer space w ith a rep eat period o f 152.3 Ä (14); (b) a single bilayer w ith a 63 Ä repeat; (c) a fluid like arran g em en t o f m yelin lipids o r o f a lipid and lipid-protein m ixture w hich p ro d u ced the intense reflection o f a b o u t 44 Ä [11]; (d) a cholesterol phase th a t p ro d u ced the 33.5 Ä series, a n d (e) the 14 Ä series, w hich m ight co rresp o n d to a form o f cholesterol [ 1 1 ]. The co m p ariso n o f the X -ray p a tte rn s obtained und er the sam e conditio n s from the sciatic nerves o f P C P treated a n d u n tre a te d rats did n o t show significant differences in th e n um ber, intensity and o rien tatio n o f th e reflections neither in th eir interp la n a r spacings. T his result could easily lead to the conclusion th a t P C P did n o t affect the stru ctu re o f
myelin. H ow ever, the observations m ade by o p ti cal and electron m icroscopy o f dry fibers indicated th a t PC P h ad indeed produced degenerative changes to som e o f the nerve fibers. T he d iscrep an cy between these results and those o b tain ed by X -ray diffraction can be explained. In fact, optical and electron m icroscopy allow ed to observe details o f single fibers. Therefore, it could be detected th a t the 10% o f the A and B type o f them show ed dege nerative changes (Fig. 3 - 5 ). O n the o th e r h and, all the fibers co ntribute to the resulting X -ray diffrac tio n patterns. Thus, the discrete reflections arise from the 90% o f intact fibers w hereas the rem ain ing 1 0 % , consisting o f structurally p e rtu rb e d and random ly distributed fibers, w ould only p ro duce a diffuse scattering. In fact, observations m ade on the X -ray diagram s o f the sciatic nerves o f PC P treated rats, b o th wet and dry, revealed a higher background th a n those obtained from the norm al rats.
Acknowledgements R esearch g rants from F O N D E C Y T (0783/88 and 0625/89) and the U niversity o f C oncepcion (20.13.79 and 20.36.02) are gratefully acknow l edged.
Table III. Assignment of the reflections obtained from dry sciatic nerves o f rats1. Double Bilayer do (Ä) Order
Single Bilayer do (Ä) Order
152.3 76.2
63.1 31.6 21.0 15.9 12.7 6.9 6.3 4.19
(1) (2)
(1) (2) (3) (4) (5) (9) (10)
Fluid Phase 43.7
Cholesterol do (Ä) Order 33.5 16.8 11.4 6.9 5.2 5.1 4.9
(1) (2) (3) (5)
14 Ä Series (do (Ä) 14.0 5.8 4.6
1 Only the reflections from dry sciatic nerves o f rats untreated with PCP are con sidered in this Table.
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