Document 44Za5ZxJ5kjx8gLERyazMk1Le

a. aw** tu Wir'd / Om-ft i Mature Vol. 2S6 August 21 197S rcgrowth) of a peripheral nerve leads to a remarkable over production of Schwann celts in the distal nerve stump reaching a point at which theseccllsarc 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 titan in normal nerve, an increase in (he 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 thcaxon-Scliwnnn cell interaction result ing in mitogcncsis illustrates one use of cultures containing either `pure' Schwann cell populations or bare ncuritc pre parations. These preparations may be useful in further analysis of the interaction between ncuritc 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 72; accepted June 16,1975. Flschbach. G. D.. Dttl Dio/.. 2S. 407 (1972). * Bunpe, ft., Jtfcs, RH Wood, P., Burton, ft., and Ko, C. *P. Brain Res., 66* 401 11974). * Murray. M., and Stout, A. P.. Am. J. Path., 16, 41 (1940). Bunpe. R. P.. and Worn!, P. M,, Brain firs., 7. 261 (1975). 9 Causey. G., The Cell of StitH-ann (Livingston. Edinburgh and London, I960). Thomas. P. k.. i.Anat., 106. 465(1970). ' Stirling. C. A.. Brain Res.. 87, 130 (1975). Logan, Rossiter, R. J.. and Barr, M. L., J. Anat,, $7,49 (1953). Biological reactivity of PVC dust Concern has been expressed about the biological potential of polyvinyl chioridc (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 technique5 which is useful for assessing the degree of membrane-induced damage by a variety of toxic materials. Other in vitro screening systems using lung3 and other cells4 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 ami biologically reactive chrysotilc asbestos A (UICC standard reference sample) and the clfcct of PVC on lung fibroblast cultures. The haemolysis technique used has been described in detail previously5. The degree of haemolysis was expressed as a percentage of the totally lysed sample and the results presented as the means ami ranges (if any) of, at least, quadruplicate assays. Two samples of PVC were tested (KM 1 and KM2) both of which were obtained ns linclv divided ilt ictl powders which had been formed by standard processing procedures for use in fabrication work. The ItrsTsample (KM 1) was highly haemolytic at relatively low concentrations. 100"' haemolysis (over 50 min) being Achieved by tie tween 7.3 and It) mg of dust (I'ig. In). With increas ing concentrations of PVC, haemolysis seems to be reduced, hut tltis cllcct is most likely the tesiilt of some of the released !f ujjJuL 80 2 60 40 'fso 20 0 10 20 30 40 50 60 70 80 0 20 40 60 80 Dust sample (mg) Time (min) Fig. 1 Haemolysis by PVC dusts and chrysolite asbestos, c. Haemolytic potency of KM I (O) and K M 2 (O) PVC samples after 50 min: b, change in haemolysis with time by 7.5 mg samples of UICC chrysotilc asbestos A (A) and KM 1 (O) PYC. Briefly, a 1 % (v/v) suspension of packed rabbit erythrocytes in veronal buffered saline, pH 7.4, was used in all me experiments. The incubation mixture normally consisted of dust sample with 1 ml I % erythrocyte suspension + 3 ml veronal bulfcrcd 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. Controls consisting of a totally lysed sample (1 ml 1 % erythrocyte suspension -} 3 ml water) and a fragility control (I ml 1% erythrocyte suspension + 3 ml veronal bulfcrcd saline) were treated identically in each experiment. haemolytic. A sample of 100 mg of KM 2 PVC gave an equiva lent haemolytic effect to 1 mg of sample KM 1. The haemolytic activity with time of a sample of KM 1 PVC (7.5 mg) was compared with an equivalent mass of chrysotilc asbestos A (Fig. 16). It is evident that the asbestos is a faster haemolytic agent, although total lysis is achieved by PVC after I h. During the processing of different forms of PVC, a variety . Fig. 2 Haemolysis by washed samples of KM I PVC dust (7.5 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 I;/, wash 2; g, wash 3. K 0> C . -gents nrc used which could contribute to the haemolytic .Teef of the dust. Thercfoic, samples (7.5 nig) of KM I I'VC were washed once, twice and thiec times (5 s each wash, in 3 ml veronal butler on a wlnrliniix; followed by centrifugation, 2,000 r.p.m. for 20 min) and the haemolytic potency or the washed dust samples and the supernatant Hu id were compared with the lytic potential of tin untreated I'VC sample (Fig. 2). After a single wash, the haemolytic potency of KM I I'VC is reduced by over (>0% 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 l'VC dust. It is also evident that this material, once removal from the dust surface and diluted out in solution, has very limited haemolytic activity (Fig. 2). The effect of KM I I'VC was studied on the levels of cell 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 the analyses for DNA, RNA protein and hydroxyproline have been detailed previously3 5, before addition of different concentrations of KM 1 PVC (50-200 pg ml'1 culture medium or 0.5-2.0 mg per culture) the dust sample was first washed in a balanced salt solution containing antibiotics (see legend to Fig. 3). Other methods of dust sterilisation (heat, autoclaving, radiation) forms of PVC dusts exhibit a high hasjxtfllyaigjvHcntial hegause of~Ttn~-piysnTrc~oT~,i readily Mil'jh(cJ_siii_f.ice .i'Suci,i(ed agent. Exposure to tins type of I'VC dust may--thus constitute an addition,il health'hazard because of the increased biological activiTy""of the dust. Work" is in progress to determine the nature ol the haemolylically-active agent by assessing a wide range of I'VC dusts of which complete knowledge has been obtained of (lie chemical processing. The possibility that VCM is (lie active agent has been explored, but both samples tested were found to contain immeasurable amounts of VCM (<l p.p.m.). Nevcrtlveless, the present study indicates that the introduction of a washing procedure after (lie processing of KM I PVC dust would certainly reduce or abolish the haemolytic activity of tin's material. We thank Miss Karen Mitchell for assistance and two of us (R.D. and l'.M.II.) thank the Medical Research Council for financial support. R. J. Richards Rasiimihala Dlsai P. M. Hexr F. A. Rose Department of Bioehemistry, University College, PO Box 78, Cardiff CH ! XL, UK Received June 2; accepted July M, 1975. ISelikofT, I. J., and Hammond. E. C.. (Ed*). Toxicity of Vinyl chloride-polyvinyl chloride, Ann. )*. Acud. Sci.. 246. 377 (1975). * Harington. J. S. Miller. K.. and MacNab. G., Environ. Res,, 4. 95*117 (1971). J Richards, R. J., ami Morris. T. G.. Life Sci.. 12. 441*451 (1973). * Alison, A.. I tarin? ton. J. S.. and Hirbcck, M. i.,J. exp. Med.. 124, 141- I6i ((Wi6). * Richards, K. and Wustemun. K. in Tissue Culture in Medical Research, 91-99 (Heincmann, London. 1974). (//g per ml culture medium) t/ig per nil culture medium) PVC concentration (mg per culture) PVC concentration (ing per culture) Fig. 3 The efiect of different concentrations of KM I PVC on lung fibroblast cultures. The PVC sample was lirst washed with an antibiotic solution (penicillin (100 units) streptomycin (ICO MB) in 1 nil) and then added as a single dose to 3-d-oMeultiircs (in logarithmic grow th). Cultures were inainlained in 10 ml of 20% foetal bovine serum plus Waymouih's medium containing additional ascorbic acid1, changed twice weekly and removed for analysis on day 24. were not considered to be feasible and while the washing pro cedure undoubtedly reduces the ability of this PVC sample (0 damage ceff membranes (Fig. 2) fibroblast cultures treated with the dust all had lower levels^ of cell mal hydroxyproline after 24 d (Pig. 3). Sonic fluctuat ion in DNA levels was apparent with dillcrcnt dus'f concentrations but the significance of these is doubtful without further ex|>erinieiilatioi). There is little change in (lie level of lolal pnucm or RNA in the PVC-trealed Cultures. From these preliminary findings we conclude that certain Mechanism of induction of haemolytic anaemia by phcnylhydrazinc A compound with the optical spectrum of a fcrrihacmochrome was produced when ferricyanidc-oxidiscd phenylhydrnzine was added to a solution of fcrrihaemoglobin*. The three isomers of methylphcnylhydrazine similarly resulted in ferrihacmochromcs, but 4-hydrazinobcnzoic acid did not3. The induction of hae molytic anaemia by a substituted phcnylhydrazinc was related to the reactivity of its oxidised form with fcrrihaemoglobin to produce a fcrrihacmochrome3,3. Further studies of the reaction of oxidised arylhydrazine with ferrihaemoglobin have established that the formation of a fcrrihaemochromc-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 phcnylhydrazinc. Substituted phcnylhydrazinc 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 recrystalliscd product were confirmed by its NMR spectrum, infrared spectrum, melting point, and analyses for C, H. N, and Cl. Oxyhacmoglobin solutions were deoxygenated by the passage of oxygen-free* nitrogen or helium, and the resulting fcrrohacmoglobin was oxidised to fcrrihaemoglobin by the addition of excess forricyanidc. To a solution of fcrrihaemo globin in excess fcrricynnide, a solution of arylhydrazine hydrochloride in water or ethanol was added, and the optical spectrum (hat resulted was recorded with the Cary Model 17 instrument. The s|>cetra obtained with 3- and 4-eliloroplienylhydra/.ine were similar to that previously reported with unsubstilutcd phciiylhydrazine'. The spectra obtained with 3,4- and 3,5-dichloropheiiylhydra/ine were distinguished by a more prominent absorption band in the red than llte others. 2-C'hloro- and 2,3-, 2,4-, and 2,5-dichlorophenylliydia/ine resulted in spectra with a single broad maximum at around 540 -550 mu and wilhout a hand in the red. 2-(i-l)iclilomphcnyl- Itydruzinc, 2,6-dimethylphcnylbydrazinc, 2-ltydrazinobui/oic 1469002 BFG14013 ITT T