Document 5bQXZYpNBbYJOn7k8aBXK8bL5

664 regrowthVof a peripheral nerve leads to a remarkable over- productidn 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 irfjury'. 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 of Anatomy, Washington University School of Medicine, St Louis, Missouri 63110 Received May 22; accepted June 16. 1973. 1 Fischbach. G. D , Deri Biol., 21. 407 (1972). 1 Bunae, R., Rees. R., Wood. P , Burton, H., and Ko, C. -P., Brain Res., 64, 401 (1974). J Murray. M.. end Stout, A. P.. Am. J. Path., 14. 41 (1940). 4 Bunge. R. P.. and Wood. P. M.. Brain Res., 57, 261 11973). 3 Cauaey, G., The Cell of SvAwtuiff (Livingston, Edinburgh and London. I960). Thomas. P. K., f. Anar., 106. 463 (3970V 7 Stirling, C. A., tram Ret.. 17. 130 < 1975). 1 Logan. J. E., Rossiter, R. J., and Barr, M. L . J. Anar., 17, 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 1 andKM2) both of which were obtained as finely-divided dried powders which had txen formed by standard processing procedures for use in fabrication work. The firsLsample (KM 1) was highly haemolytic at relatively low concentrations,' l(X^% haemolysis (over SO min) being achjeved by between 7?3and iCthioT dust (hg. Id). With iuueising concentrations ot rvc, 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 PVC wus found to he practically non- Xature Vol. 256 August 21 li?5 Va-ure I, of agenu effect of were wa.hc. 80 3 ml verona1 2,000 r.p m 60 washed dusi with the lyti After a sing1 " 40 reduced by actixJuy r 20 V surface-avvo, soluble form this PVC dufrom the du 0 10 20 30 40 50 60 70 80 0 20 40 60 limited haem Dust sample (mg) Time (min) The effect DNA, RNa Fig. 1 Haemolysis by PVC dusts and chrysotile asbestos.''' a. Haemolytic potency of KM 1 (O)and KM 2(#) PVC samples", after SO min; b, change in haemolysis with time by 7.5 mg samples , of UICC chrysotile asbestos A (A) awl KM 1 () PVC. ' Briefly, a I */, (v/v) suspension of packed rabbit erythrocytes ia * veronal buffered saline, pH 7.4, was used in all the experiments.1 The incubation mixture normally consisted of dust sample with J 1 ml I % erythrocyte suspension + 3 ml veronal buffered saline., After incubation and agitation for 50 min (or varying time inter-. vals) at 37 CC the samples were centrifuged at 2,000 r.p.m. for ` 20 min and the extinction of the supernatant rod at 541 nm.2 Controls consisting of a totally lysed sampled ml 1 % erythrocyte suspension *3 ml water) and a fragility control (1 ml l'/,- erythrocyte suspension + 3 ml veronal buffered saline) were ^ treated identically* in each experiment. * . : ,fi haemolytic. A sample of 100 mg of KM 2 PVC gave an equive-; lent haemolytic effect to 1 mg of sample KM I. The haemolytic j collagen) in I The methods DNA, RNA previously* *. KM 1 PVC 1 per culture) i salt solution l methods of i 24, 22 \ 20 L \ activity with time of a sample of KM 1 PVC (7.5 mg) wu compared with an equivalent mass of chrysotile asbestos A (Fig. 16). It is evident that the asbestos is a faster haemolytic | agent, although total lysis is achieved by PVC after 1 h. During the processing of different forms of PVC, a variety j *= r >&, ok . o> X isL I6i 141 0 Fig. 2 Haemolysis by washed samples of KM 1 PVC dust (73 mg) and the resulting supernatant fluids from these washingi*j compared with the haemolytic activity of the untreated dust' pvc a i/ig per sample. KM I PVC a. Untreated; 6, washed once; c. washed twice; d, washed three times; e, wash 1; /, wash 2; g, wash 3.. 2.8- r 100 r- 2.6 80 60 L 40 L T T 20 L k E 2.2 DTH -4 000043438 " 2.0_________ ii PVC ' in;. FI*. 3 Theefi ! v lung fibroblast an antibiotic . Uf) >n 1 ml) an. > logarithmic gn> ? foetal bovine additional asc," 4. -r . were not considi cedure undoubu to damage cell r with the dust al after 24 d (Fig. 3. with different du b doubtful witt change in the lev, cultures. ; From these pi \atu~e i'oi .-lai-aw -1 IV/5 665 of agents are used which could contribute to the haemolytic effect of IhjT&iAi. Therefore, samples (7.5 mg) of KM 1 PVC forms of PVC dusts exhibit a high haemolytic potential because of the presence of a readily soluble, surface-associated agent. ere washed once, twice and three times (5 s each wash, in 5 ml veronal buffer on a whirlimix; followed by centrifugation, 2,000 r.p.m. for 20 min) and the haemolytic potency of the ashed 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 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 reduced by over 60 a{ and subsequent washes reduce the activity of the dust even further. Thus the removal of some nirface-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 is the active agent has been explored, but both samples tested were found to contain immeasurable amounts of VCM (<l 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. ".I limited haemolytic activity (Fig. 2). .......... .. . ... ,t . ! The effect of KM 1 PVC was studied on thelevels ofcell mat DNA, RNA, protein and hydroxyproline (assessment of ' collagen) in lung fibroblast cultures maintained in vitro for 24 d. 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 . The methods of isolation and cell culture and the analyses foe. r DNA, RNA protein and hydroxyproline have Tseen detailed 7 previously'**. Before addition of different concentrations of > -v-' Rashmibala DeJai P. M. Hext _ F. A. Rose 1. KM 1. PVC (50-200 pg ml*1 culture medium or 0.5-2.0 mg Department ofBiochemistry, .< per culture) the dust sample was first washed-in a balanced , University College, PO Box 78, > * alt solution containing antibiotics (see legend to Fig. "3), Other CardiffCFU XL, UK V methods of dust sterilisation (heat, autoclaving, radiation) * Received June 2; accepted July 14, 1975. '*%t& i2 1 t's. V$ rl ..'3l i* V0 *1v:X 24 22 \ \" r =` * 20 v' \T = 50- w 1\ r _ ' its N*---- a. 40f J, 5. ' L 16 *< 1 Z 30 - 2. 14 o -j------ioo - - 20 :w * Selikoff, !. J.. and Hammond, E. C.. (Eds). Toxicity of Vinyl chtoeide-Potyrmyt chloride, Ann. td.Y. Acad. Sci., 246. 377 (1975). * 2 Harmiton, J. S , Sftiler. K.. and MacNab, G., Environ. Ref.. 4, 95-117 (1971). > Richards. R. J . and Morns. T. G . Life Sci.. 12, 44 MSI (1973). Allison. A Hanngion. J. S .and Birbeck. M i , J.exp Med , 124.14M6I (1966). 5 Richards. R. J.. and Wusteman. F. S., in Tissue Culture in Medical Research, 91*99 (Hcincmann, London. 1974). Mechanism of induction of haemolytic anaemia by phenylhvdrazine : A compound with the optical spectrum of a ferrihaemochrome .100 200 was produced when ferricyanide-oxidised phenylhydrazine was PVC (km 11 concentration ", PVCJ_concentration _ (/<g per ml culture medium) J (/Sg'pcr mFcnlture medium) ^ added to a solution of ferrihaemoglobinT The three isomers of methylphenylhydrazine similarly rewltedinfeiTihaemochromes, but 4-hydrazinobenzoic acid did not1. .TKe induction of hae molytic anaemia by a substituted phenylhydrazine was related to the reactivity of its oxidised form with ferrihaemoglobin to' produce a ferrihaemochrome1-'. Further studies of the V* w* r! vs If reaction of oxidised.arylhydrazine with ferrihaemoglobin have established that the formation of a ferrihaemochrome-1 ike product and the character of the optical spectrum of this product depend on the nature and position of substituents . on the benzene ting of- phenylhydrazine. . Substituted phenylhydrazine hydrochlorides were obtained 4t lire* 2U0 ,. ;U,, .; .-...Wa*-,- 200. from commercial sources or were synthesised by standard ,2. PVC concentration .... ..PVC cuncaitrattou procedures. Each compound was purified by recrystallisation (mg pci eulmi.) ,*. (nig per culture)', from 2 N HCI or ethanol, and the structure and purity of the J Fig. 3 The effect of different concentrations of KM 1 PVC on I lung fibroblast cultures. The PVC sample was first washed with ! m antibiotic solution (penicillin (100 units) streptomycin(100 J Mg) in I ml) and then added as a single dose to 3-d-old cultures l in i logarithmic growth). Cultures were maintained in 10 ml of 20% 5 ft*! bovine serum plus Waymoulh's medium containing . ] additional ascorbic acid1, changed twice weekly and removed for analysis on day 24. recrystallised product were confirmed by its NMR spectrum, infrared spectrum, melting point, and analyses for C, H. N, jnd Cl. Oxyhaemoglobin solutions were deoxygenated by the passage of oxygen-free4 nitrogen or helium, and the resulting errohaemoglobm was oxidised to ferrihaemoglobin by the addition of excess fcrricy.mide. To a solution of ferrihaemozlohm in excess ferricyanide. a solution of arylhydrazine vre n-t considered to be feasible and while the washing pro- ~-1"* 11 " !|v reduces the ability of this PVC sample m1' ntes 11 iy. 2) fibroblast cultures treated itn ttie o..-: lower levels of cell mat hydroxyproline Jafter24 d (Fig. 3). noi ihicvuntii'n in DNA levels was apparent t tith different dust cr (rations but the significance of these doubtful without l-* (tenge in the level of cultures. i experimentation. There is little protein or RNA in the PVC-trealed From, these prelim: findings we conclude that certain h>dr.'chloride in water or ethanol was added, and the optical >f-v:(rum that resulted was recorded with the Cary Model 17 instrument. The spectra obtained with 3- and 4-chlorophenyl- Indra/inc were similar to that previously reported with un- 'ubsimued phc-nvlhydrazine1. The spectra obtained with 3.4- .rid 3.5-dichluroplienyIhydra/ine were distinguished bv a m,ml ,imminent absorption band in the red than the other- 2-< In.no- and 2.3-, 2.4-. and 2,5-dichloroplienylhydrazi re-ulied hi spectra with a single broad maximum at aroun.' San so nm anj milioui a bund in the red. 2-6-Dicliloropheny I- (mil , I'll- in tliv Iplienv lliydru/inc, 2-lndra/ir DTH 000043439