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Cytotoxicity of Heated Chrysotile by Hisato Hayashi* Ertvirntmental Health Perspectivej VoL 3. pp. Jbr-e.'O, I97i Cytotoxicity and hemolysis were studied in chrysotiie and quarts. The biological activity of the surface seemed to be different between chrysotile and quarts. Quartz lost its cytotoxicity on heating over about 300*C. However chrysotile showed remarkable toxicity aod induced hemolysis on heating between Hafl and agQ'*C. compared the original unheated soecimeol. iSie mice injected iotraperitoneallv with minerals bested in this temperature range den^ni;'-- died within 43 hr after injection, while those injected with untreated chrysotile or chrysotile heated in -the other heat ranges did not. The products in this range were highly disordered materials. It was assumed that the change of biological effects resulting from heat treatment may be related to the disordered state of chrysotile in the process of transformation into forsterite. The relationship between chemical character and cytotoxicity of the heated chrysotile specimens was also studied. We have studied the cytotoxicity of mineral dusts.to clarify the pathogenesis of pneumoconiosis. We found that cytotoxicity and the ability to induce hemolysis depend on sur face properties of the minerals' dusts. As shown in Figure 1, alkali-treated quartz showed in creased acid phosphatase activity. This quartz has a strong cytotoxicity, but it did not, however, induce striking hemolysis. On the other hand, although ground quartz was weakly cytotoxic, it easily induced hemolysis. The chrysotile has strong cytotoxicity as well as a strong ability to induce hemolysis. Both chrysotile and amphibole can induce hemolysis, and they have a strong cytotoxic effect on the macrophage. Therefore, there is a difference between the quartz and asbestos, with respect to hemotysis and cytotoxicity. Chrysotile was heated at various temperatures for 1 hr, and then we examined biological effects of heated chrysotiie deter mining the degree of lactic acid production and hemolysis. Hemolysis induced by chrysotile was "National Inst.it-.. of Industrial Health. Kawasaki. Japan. Present ace -*: Mining College. Akita University. Akita. 010. Japan. Hemolysis Acid phosphatase activity *tiPCv-oo ct 533nip Extinction at SOS mjj 0.5 ________ 0_______ 0.5 -AikcU Aikdi ] Ground qucrtz (<0.5 u) [ Ground quart: (0.5- 2 p) 4=3 Control (without dust) -c:ed .cucrtz EffiKKEESSsSaSSSBSSa (<0.5 u) cted ever:: (15-2 y) P"CVia. I. He--oiysis and acid phosphatase activation as sociated with c-carra particles. striking after heating in the temperature range 500-- SOO'C iFig. 2). On the other hand, lactic acid production increased until 500C. de creased abruptly until 650^0, and then con tinued to increase with increasing temperature of heating (Fig. 2). On DTA. chrysotile has an endothermic peak followed by an exothermic peak. X-rav and in frared analysis proved that chrysotiie was rnn- ,r.:o : ^rsterice on heating to 500--.700G 5:r.ce c.nrysotiie and torsterite coexist in this range, the rapid changes in the biological activi ty of chrysotile may be caused by the highly dis ordered condition during the transformation. December 1974 267 FIGURE 2. Cytotoxicity of chrysotile (UICC) treated at various temperatures. Both the unheated sample and the sample heated at 650C had a very high suppressing ef fect on the lactic acid production. However, when 0.2 ml of 10% bovine albumin was added to the cell-dust system, the two samples showed a different behavior. The inhibitory effect of the unheated sample was remarkably reduced by presence of albumin, but the effect of the sam ple heated at 650C was not reduced (Table 1). A similar phenomenon was also observed in hemolysis. Hemolysis induced by the unheated sample was reduced by 50% by the addition of 11 fig of albumin, but in the case of chrysotile heated at 750*C, 4000 fig of albumin was necessary to obtain the same reducing effect This means the unheated sample can easily ab sorb protein and become inert. However, ab sorption of protein was difficult in the heated samples. We found chrvstntilp hpatpd h*fwfnt 650C and 750C to be very toxic. . This fact was confirmed by animal ex periments with intraperitoneal injection of this chrysotile (Table 1). The mean gain in body weight 8 days after injection reflects this. It was very significant that all mice which were in jected with chrysotile heated at 65^ or 750C died within 2 days after injection. Further experimental studies were made to clarify the relationship between chemical character 2nd cytotoxicity. Chrysotile was heated at various temperatures for 1 hr, then 10 mg of each sample put in water and agitated for 4 hr/day at 25C. A supernatant was obtained by centrifugal separation, and the pH and dis solved amounts of Si and Mg in the supernatant Table I. Effects of heat treatment of chrysotile (UICC) od effects on macrophages, erthryocvtes, and weight gain in mice. Heat treatment temperature, c Lactic acid production without albumin, of control Lactic acid production with albumin, tr of control Hemolysis, tc of control Amount of albumin required to prevent hemolysis by 50*T. Mg Cain of body weight 8 days after injection,g Untreated 30 112 52 11 8.0 200 4S 300 66 26 36 n 3S 7.2 500 90 12 21 7.7 650 25 36 ST 750 5S 44 650 73 15 1000 125 6 9000 4000 540 10 Died Died 64 5.1 26S Environmental Health Perspectives &r lt c,re determined. This procedure was repeated Figure 6 shows data obtained from superna ' on successive days for 15 days. The pH value of tant in the experiment in the first day. The the supernatant of the samples heated at'650 and 750aC was higher than that of the other samples (Fig. 3). Amounts of dissolved Si and Mg in the supernatant of this group were also larger (Figs. 4 and 5). T3 3 3. Amount of dissolved Si in supernatant as a function of temperature of heating of chrysotile: ' < unheated: (Q) 500*C: (o) 630*C; (A) 750'C; () 1000*C. cn 2 *9 o 150 :n c 3 o 100 a a 50 Gj > o CO 0 I 2 3 4 5 6 7 8 9101! '2131415 uo a Days * 4. Amount of dissolved Mz in supernatant as a action of temper 'ure of heating of chrysociie: ' unhealed; ( 501. (o) S50aC; (a) T50*C. fl 1000'C. C a a c w_ o CL 3 7-8^A_ in 74- 0 1^"- 70: a, Q. 1 2 3 4 5 6 7 8 91011 12131415 Days FrGCSE 5. pH of supernatant as a function of temperature of heating of cirysoriie: rxi unheated; (Q) 500C; (o) 630*C. (A) 750'C: l'3`C. Nature of supernatant fluid in the first day Fracas 6 N'arjre rl supernatant fluid as a function of temperature ."rav.r.sr. pH; (o) Si; Mg. All data obtained on - i-v. 1 >n n p n i c^ December 1974 269 supernatant from chrysotile samples heated at 650 and 750C show higher pH and larger amounts of dissolved Si and Mg (Fig. 6). The effect of each sample on macrophage was estimated by incorporation of **C-leucine into cell protein. When the pH of the suspension and/or supernatant fluid were adjusted at 7.8, the effect on macrophage were nearly same. When we did not adjust the pH value, the data of the effect on macrophage was not constant. This means that incorporation of "C-leucine into macrophages depends on the pH of the solu tion. The differences in pH may be caused by differences in surface properties of the samples. However hemolysis due to chrysotile heated a; 6503C did not depend on pH of the solutions. i l 3 3 & i 'j 270 Environmental Health Perspective?