Document G5aaOJQxELe9mra754eoKo7p7

*6A regrowth)/of a peripheral nerve leads id a remarkable over- produciidn of Schwann cells in the distal nerve stump reaching a point at which these cells are applied in multiple layers around each single axon. Also, in crush lesions of the abnormal peripheral nerve roots of dystrophic mice, where a paucity of Schwann cells and myelin segments provides much less degen erating material than in normal nerve, an increase in the number of myelin segments and, presumably, Schwann cells is induced by crush injury'. The importance of the axon in provoking Schwann cell proliferation is also indicated by observations, on the proximal stump of severed or crushed sciatic nerve, that proliferation occurs independently of widespread nerve fibre or myelin degeneration and in con ditions in which nerve regeneration is most actively occurring*. Our observation of the axon-Schwann cell interaction result ing in mitogenesis illustrates one use of cultures containing either `pure' Schwann cell populations or bare neurite pre parations. These preparations may be useful in further analysis of the interaction between neurite and Schwann cell, as well as in studies of the basic properties of the Schwann cell itself. This work was supported by the National Multiple Sclerosis Society. Patrick M. Wood Richard P. Bunge Department ofAnatomy, Washington University School of Medicine, St Louis, Missouri 63110 Received My 22; accepted June 16, 1975. ' Fischbich, G. D.. Dfl Biol., It. 407 (1972). " 1 Bunge, R,, Rees. R,, Wood. P.. Burton, H,, and Ko, C. -P., Brain Bn.. 66, 401 (1974). 5 Murray, M., and Stout, A. P,, Am. J. Path., 16. 41 (1940). 4 Bunge, R. P- and Wood, P. M., Brain Bet., 57, 261 (1973). 1 Causey, G., The Cellof Schwann (Lmngston. Edinburgh and London. I960). Thomas, P. R... I. Anar., 104, 463 0 970), ' Sorting. C. A., tram Bn.. S7, 130 (1975). Logan, ., Rossiter, R J.. and Barr, M. L,, J. Anar., *7,419(1953). Biological reactivity of PVC dust Concern has been expressed about-the biological potential of polyvinyl chloride (PVC) and its associated monomer vinyl chloride monomer (VCM)1. During the industrial processing of PVC, workers can be exposed to varying quantities of this material in the form of a dust. At the present time, however, there is little detailed biological or biochemical information on the effects of inhaled or ingested PVC. or on the reactivity of this dust material. The biological reactivity of other dusts (silica, asbestos) have been studied by a haemolysis technique* which is useful for assessing the degree of membrane-induced damage by a variety of toxic materials. Other in vitro screening systems using lung* and other cells* have also provided useful information on structural and biochemical changes induced by particulate matter. Here we report on the haemolytic potential of PVC dust, comparison of which is made with the highly haemolytic and biologically reactive chrysotile asbestos A (U1CC standard reference sample) and the effect of PVC on lung fibroblast cultures. The haemolysis technique used has been described in detail previously*. The degree of haemolysis was expressed as a percentage of the totally lysed sample and the results presented as the means and ranges (if any) of, at least, quadruplicate assays. Two samples of PVC were tested (KM I and KM2) both of which were obtained as finely-divided dried powders which had txsn formed by standard processing procedures for use in fabrication work. The fksLsarmple (KM 1) was highly haemolytic at relatively low concentrations,- 100% haemolysis (over 50 min) being achieved by between 7,5 and lOmgofdusi (hg. Id). With tnuciiying concentrations of PVC, haemolysis seems to be reduced, but this effect is most likely the result of some of the released haemoglobin binding to the dust, which is then spun down into the pellet- Sample KM 2 I'VC was found to he practically non- Xature Vol. 256 August 21 1925 aiure ) - 100 r ` of agents a-1 effect of : 80 60 * 40 20 k u 0 10 20 30 40 50 60 70 80 0 were wa ,hc, 3 ml veronal 1 2,000 r.p.m washed dust with the lyti After a sing! reduced by actirjy tni t* surface-as'.o, soluble form this PVC dii' from the do limited haem Dust sample (mg) Time (min) The effect; DNA, RSA| Fig. 1 Haemolysis by PVC dusts and chrysotile asbestos. '' a. Haemolytic potency of KM 1 (O)and KM 2 () PVC samples after 50 min; b, change in haemolysis with time by 7.5 mg samples of UICC chrysotile asbestos A (A) and KM 1 () PVC. ' Briefly, a 1 % (v/v) suspension of packed rabbit erythrocytes in * veronal buffered saline, pH 7.4, was used in all the experiments.J The incubation mixture normally consisted of dust sample with > l ml 1 % erythrocyte suspension+ 3 ml veronal buffered saline., After incubation and agitation for 50 min (or varying time inter-. vals) at 37 C the samples were centrifuged at 2,000 r.p.m. for 20 min and the extinction of the supernatant read at 541 nm.1 Controls consisting of a totally lysed sample (I ml I%erythrocyte suspension 4-3 ml water) and a fragility control (1 mil'/,-. erythrocyte suspension + 3 ml veronal buffered saline) wett^ treated identically in each experiment. f 1 collagen) in I The method;, DNA, RNA previously' ` KM 1 PVC per culture) 11 salt solution c methods of 24 3 "1 3 O \u * haemolytic. A sample of 100 mg of KM 2 PVC gave an equiva 5 20 b \ lent haemolytic effect to I mg of sample KM 1. The haemolytic j k-r activity with time of a sample of KM 1 PVC (7.5 mg) w compared with an equivalent mass of chrysotile asbestos A (Fig. 16). It is evident that (he asbestos is a faster haemolytic j agent, although total lysis is achieved by PVC after 1 h. *a *t 0 km During the processing of different forms of PVC, a variety *3 >5 *X Fig. 2 Haemolysis by washed samples of KM I PVC dust (7.J mg) and the resulting supernatant fluids from these washings] compared with the haemolytic activity of the untreated dust ~ sample. KM 1 PVC a. Untreated; b, washed once; c, washed twice; d, washed three times; e, wash 1; /, wash 2; g, wash 3.- !8 I6;. 14 0 PVCtk 0'E Per 100 r- r 80 60 _> c E 40 L 20 Mi S .o - " Mil____ it pvt 5 irit Fig. 3 The efl lung fibroblast . an antibiotic v. jig) in I ml) an. 1 'j, logarithmic grtJ* foetal bovine t~ additional av 4* r .were not considi eedure undoubt, to damage cell r with the dust al after 24 d (Fig. 3< with different du b doubtful witl change in the lev, cultures. ; From these pi DTH 000042450 Voi. l,'6 2! 197$ 665 of agents ate used which could contribute to the haemolytic effect of ttydu(t. Therefore, samples (7.5 mg) of KM 1 PVC were washed once, twice and three times (5 s each wash, in * 3 ml veronal buffer on a whirlimix; followed by centrifugation, * 2,000 r,p.m. for 20 min) and the haemolytic potency of the i washed dust samples and the supernatant fluid were compared * with the lytic potential of an untreated PVC sample (Fig. 2). - After a single wash, the haemolytic potency of KM 1 PVC is * reduced by over 60 and subsequent washes reduce the ! activity of the dust even further. Thus the removal of some ! surface-associated material, which must exist in a reasonably * soluble form, considerably reduces the biological potency of this PVC dust. It is also evident that this material, once removed : from the dust surface and diluted out in solution, has very limited haemolytic activity (Fig. 2). . , . ,t..... . ' The effect of KM 1 PVC was studied on the'levels ofcell mat DNA, RNA, protein and hydroxyproline (assessment of * collagen) in lung fibroblast cultures maintained in vitro for 24 d. The methods of isolation and cell culture and Ihc analyses for. r DNA, RNA protein and hydroxyproline have Tseen detail$d| * previously* *. Before addition of different concentrations of * KM l.PVC (50-200 pg ml'1 culture medium or 0.5-2.0 mg ; per culture) the dust sample was first washed4n a balanced 7 jilt solution containing antibiotics (see legend to Fig. 3), Other - methods of dust sterilisation (heat, autoclaving, radiation) * t *2 .* 3 si r ' 22 \ > 60r 20 - XT 1 5o: forms of PVC dusts exhibit a high haemolytic potential because of the presence of a readily soluble, surface-associated agent. Exposure to this type of PVC dust may thus constitute an additional health hazard because of the increased biological activity of the dust. Work is in progress to determine the nature of the haemolytically-active agent by assessing a wide range of PVC dusts of which complete knowledge has been obtained of the chemical processing. The possibility that VCM is the active agent has been explored, but both samples tested were found to contain immeasurable amounts of VCM (<1 p.p.m). Nevertheless, the present study indicates that the introduction of a washing procedure after the processing of KM 1 PVC dust would certainly reduce or abolish the haemolytic activity of this material. _ We thank Miss Karen Mitchell for assistance and two of us (R.D. and P.M.H.) thank the Medical Research Council for financial support.' ' R. J. Richards Rashmibala Desai P. M. Hext F. A. Rose 1. ' Department ofBiochemistry, , University College, PO Box 78, Cardiff CF! I XL, UK Received June 2; accepted July 14, 1975. * 1 Selikoff, I. J., and Hammond. E. C., (Eds). Toxicity of Vinyl chlortde-Polyvinyl chloride, Ann. N.Y. Acad. Set., 244. 377 (197)). 2 Hirmgton, J. S., Miller, K... and MacNab, G,, Environ. Res.,4, 95-117 (1971). 1 Richards, R. J., and Morris, T. G., Life Sn., 12. 441-431 (1973). 4 Allison. A . Haringion. J. S . and Birbeck, M. J.,/. exp Med., 124.141-161 (1966). ' Richards. R. J., and Wusteman, F. S., in Tissue Culture in Medical Research. 91-99 (Heinemann, London. 1974). ;v-j Ny.I ; - - r |: ?.'S tl 18 i'S\. T i S=* 40T V'o 16 * < -* Z 30 2- i-x 14 o j_______________ . 20 . ioo - :<jo o .100 200 PVC (km I) concentration PV^copcenimijon . '{/<g per ml culture mediumi " ySg'pir mhcqlture medium) _ a 2.8 2.6 2.4 e= -nvz u *V 4;~ ,4. 2.2 2.t)i 1) 10(1 PVC eoneenli jtiou (mg pur culiuic) .. ;U,. . JUU:-.- . 200- , , ..PVC vuncentratiou (riig per culture)', I Rg. 3 The effect of different concentrations of KM 1 PVC on 1 lung fibroblast cultures. The PVC sample was first washed with f' an antibiotic solution (penicillin (100 units) streptomycin (100 j Ul) in I ml) and then added as a single dose to 3-d-old cultures (in * logarithmic grourth). Cultures were maintained in 10 ml of 20o ? foeral bovine serum plus Waymouth's medium containing additional ascorbic acid1, changed twice weekly and removed for Sf analysis on day 24. y rre m>t considered to be feasible and while the washing pro- --v" 11 ' '''*- Hv reduces li e ability of this PVC sample ' o''aiies (I ic. 2) libroblast cultures treated +*ith the 1 ' lower levels of cell mat hydroxyproline - after24 d (Fig. 3). hot tbiciuation in DNA levels was apparent ^ with different dust cr .rations hut the significance of these doubtful without thange in the level of i experimentation. There is little protein or RNA in the PVC-trcated allures. From- these prelim; findings we conclude that certain Mechanism of induction of haemolytic anaemia by phenylhydrazine A compound with the optical spectrum of a fcrrihaemochrome was produced when ferricyanide-oxidised phenylhydrazine was added to a solution of ferrihaemoglobin*. The three isomers of methylphenylhydrazine similarly resulted in JetTihaqmochromes, but 4-hydrazinobenzdtg aqid did not?.-XHe induction of hae molytic anaemia by a substituted phenylhydrazine was related to the reactivity of its oxidised form with ferrihaemoglobin to produce a ferrihaemochrome*'5. Further studies of the reaction of oxidised.arylhydrazine with ferrihaemoglobin have established that the formation of a ferrihaemochrome-like product and the character of the optical spectrum of this product depend on the nature and position of substituents . on the benzene ring of-phenylhydrazine. Substituted phenylhydrazine hydrochlorides were obtained from commercial sources or were synthesised by standard procedures. Each compound was purified by recrystallisation from 2 N HCI or ethanol, and the structure and purity of the recrystallised product were confirmed by its NMR spectrum, infrared spectrum, melting point, and analyses for C, H. N, and Cl. Oxyhaemoglobin solutions were deoxygenated by the passage of oxygen-free4 nitrogen or helium, and the resulting icrrohaemoglobin was oxidised to ferrihaemoglobin by the . ddiiion of excess ferricyanide. To a solution of ferrihaemoilohin in excess ferricyanide. a solution of arylhydrazine hydrochloride in water or ethanol was added, and the optical sp.vt/um that resulted was recorded with the Cary Model 17 instrument. The spectra obtained with 3- and 4-chlorophenylludrn/inc were similar to that previously reported with unsuhsiitutcd phenylhydrazine1. The spectra obtained with 3.4- j-ui 3.5-dichlurophcnyIhydra/ine were distinguished by a i liac prominent absorption band in the red than the other2-< lii-uo- and 2.3-. 2.4-, and 2,5-dichloroplicnylhydrazi re-ulicd in spectra wiili a single broad maximum at aroun.' 54<* 'so mu and w ithoui a band in the red. 2-6-DichlorophenylIn.li... mi-. 2/> diiitt-lhylpliciwlliyilr.i/inc. ?-li\dra/inohen/< > I,TH 0000424.51