Document g2kvmMxk7M51DY6mnymOGM7K3

R&S 115583 c l/4 regrowth! of a peripheral nerve leads to a remarkable over production of Schwann cells in the diMal nerve stump reaching a point a w hich 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 *ter\e, that proliferation occurs independently of widespread nerve fibre or myelin degeneration anil 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. Bunch Department of Anatomy, Washington University School of Medicine, St Louis, Missouri 63110 `tcucivcU May 22: tecep'ed June 16, 1975. ' FitohbjwY I\. 0../ H ot,. 2*. 407 2 Nun^e, R . R . \\ i*oJ P.. ll'irion. H.. Kot C, -P-, Bruin 66. 401 11974) 1 Murray. ,\1,, and Seoul, A. P.. Am. J. Fulfil 16. 41 (19401. 4 Dun^c. R. P.. and Wood. P. M,, Brum H<`t.. 57. 261 (1973), * Causey. * Vic Cell of *ithwunn (Li* tn^vion, I.Jjiihurgh and London, i960). Thomas, i*. K... A nut. 106. 46.) * Stirling. C A., tfruin Rrt,, 87. I JO <1975). ' Logan. J. F... Runner. R. J.. and Barr, M. L., J. Artut., 87,419 (1955L Biological reactivity of PVC dust ''oncers has been expressed about the biological potential of polyvinyl chloride IHVand its associated monomer vinyl chloride monomer (VCM)1. During the industrial processing T 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 -he effects of inhaled or ingested PVC, or on the reactivity of this I^HBnaterial. The biological reactivity of other dusts MlicaTasoestos) have been studied by a haemolysis technique1 which is useful for assessing the degree of membrane-induced damage by a variety of toxic materials. Other in vitro screening systems using lung1 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 and biologically reactive chrysotile asbestos A (UICC standard reference sample) and the effect of PVC on tung 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 tssays. Two samples of PVC were tested (KM I and KM2) bom of which were obtained as finely-divided dried powders which had been formed by s'.andard processing procedures for use in fabrication work. The firs: sample fKM I) was highly haemolytic at relatively low concentrations, 100,haemolysis (over 50 min) beiny ichieved by between 7.5 and lOnigoldust (Fig.!). With increus iny concentrations of PVC, Iniemolvsis seems to be reduced "ut this elfect is most likely the result of some of the released haemoglobin bind ng to the dust, which is then spun down im * the pellet Sample KM 2 PVC was found to be practically nou Nature Vul. J.'O August 21 IV73 Dust sample t mg i Time (min) Fig. 1 Haemolysis by PVC dusts and chrysotile asbestos. a. Haemolytic potency of KM I (;/) and KM 2 () PVC samples after 50 min; h, change in haemolysis with time by 7.5 mg samples of UICC chrysotile asbestos A (.'.) and KM 1 () PVC, Briefly, a I % (x/v) suspension of packed rabbit erythrocytes in veronal buffered saline. />H 7.4, was used in all the experiments. The incubation ini.xtt re normally consisted of dust sample with I ml 1 % erythrocyte suspension r-3 ml veronal buffered saline. After incubation and agitation for 50 min (or varying time inter vals) at 37 C the samples were cemrifuged 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 I erythrocyte suspension --3 ml water) and a fragility control (I ml I erythrocyte suspension - 3 ml veronal buffered 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 I. The haemoly tic activity with time of a sample of KM I PVC (7.5 mg' was compared with an equivalent mass of chrysoiileasbestos A (Fig. 1A). It is eviden' that the asbestos is a faster haemolyti agent, although total lysis is achieved by PVC after 1 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 tiremolytic activity of the untreated dusi sample. KM I PV< u. Untreated; b, washed onee; e, washed twice; d, washed 'hre^ times; e, wash 1; /, wa-.h 2; g. wash 3. 100 r- H a so L 40 Suture l'(</. '56 August /V.'j of iiv,cuts arc used which could contribute to the haemolytic effect of the dust. Therefore, samples (7.5 mg) of KM I PVC Acre washed once, twice and three times <5 s each wash, in 3 ml veronal buffer on a whirlimix; followed by centrifugation, 2.IKK) r.p.m. for 20 min) and the haemolytic potency of the 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 I 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 solub'c 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>, The effect of KM 1 PVC 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 previously*-1. 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) Z 24 22 5 3* 20 V -f IN i 16 - 14 t//g per ml culture mediumi (fig per ml culture medium) 2.x, 22u. 2-6;1 \t 2.4 L = ;imh 1NU-- -L It.ul . . _____ i 140. p 1UII ZOO II PVC com:cmr.i inm (me pci cullui .) __ _______________i IU0 2W PVC cuiicemralioii (mg per eullurcl Fir. 3 The effect of different concentrations of KM I PVC on lung fibroblast cultures, rite PVC sample was first washed with an antibiotic solution (penicillin < 100 units) streptomycin (100 Ug) in 1 ml) and then added as a single dose to 3-d-old cultures (in logarithmic growth). Cultures were maintained in It) ml of 20% foetal bovine serum plus Waymoiitlfs 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 to damage cell membranes (Fig. 2) fibroblast cultures treated with the dust all had lower levels of cell mat hydroxyproline after 24 d (Fig. 3). Some fluctuation in DNA levels was apparent with different dust concentrations hut the significance of these is doubtful without further experimentation. There is little change in the level of total protein or RNA in the PVC-treaiei' cultures. From these preliminary findings we conclude that certain 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<1 p.p.m.). Nevertheless, the present study indicates that the introduction of a washing procedure after the processing of KM I 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 Rashmiuala DtSAt P. M. Hext F. A. Ruse Department ofBiochemistry, University College, PO Box 78, CurdiffCFi I XL, UK Received June 2; accepted July 14, 1975. 1 SelikolV, I. J,. and Hammond, E. C,, < Ldi), Toxicity t>f Vinyl i htoritle -Polyvmvl trltloritli\ Ann, .V. Y, AcaJ. 246, .177 (1975). 1 Hanngton, J, S,, Miller. K.. and Ci., knvinm* Res.* 4, 95*M7 (197| I. - Richards, K. J., i.rul Moms. 1. IVi., 12. 441-45! (197.1). 4 AIIimir. a.. Ilunngton, J. S-aoi* liirttcck. M, J..J. exp, \hU.t 124,141-161 (I9fc6). 9 Richards. R. J., and VVuftffnwrv K S,, in Tt\sue Culture m Wuiuui Kcwutek, 91-99 (Heinemann, London *'*74) Mechanism of induction of haemolytic anaemia by phenylhydrazine A compound with the optical spectrum of a fcrrihaemochromc was produced when ferricyanidc-oxidiscd phenylhydrazine was added to a solution of ferrihacmoglohin1. The three isomers of methylphcnylhydrazine similarly resulted in ferrihaemochromes. but 4-hydrazinobenzoic acid did not1. The induction of hae molytic anaemia by a substituted phenylhydrazine was related to the reactivity of its oxidised form with ferrihacmoglohin to produce a ferrihacmochrome**. Further studies of the reaction of oxidised arylhvdrazine with ferrihaemoglobin have established that the formation of a ferrihacmochromc-likc product and the character of the optical spectrum of this product depend on the nature and position cf 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 rccrystallisation 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. Oxyhaemoglobtn solutions were deoxygenated by the passage of oxygen-free' nitrogen or helium, and the resulting fcrrohacmoglobin was oxidised to ferrihaemoglobin by the addition of excess ferricyanide. To a solution of ferrihaemo globin in excess ferricyanide, a solution of arylhydnt/ine hydrochloride in water or ethanol was added, and the optical spectrum that resulted was recorded with the Cary Model 17 instrument. The spectra obtained with 3- and 4-chlorophcn\lhydrazine were similar to that previously reported with unsubstituted phenylhydrazine1. The spectra obtained with 3,4- and 3,5-dichlorophenylhydrazine were distinguished by a more prominent absorption band in the red than the others. 2-Chloro, and 2,3-, 2,4-, and 2,*5-dichIorophcnylhydra/ine resulted in spectra with a single broad maximum at around 540-550 nm and without a band in the red. 2-6-Diehlorophcnylhydrazine, 2,6-dimethylphenylhydrazine, 2-hydrazinoben/oic R&S 115582 "\