Document e7YwEL230w66dyR6p8gOarLgG

Biochemical changes caused by asbestos dust in the lungs of rats by QAMAR RAHMAN, PhD., M.U. BEG, Ph.D., P.N. VISWANATHAN, Ph-D., and SR. ZAIDI, PhJD.1 RAHMAN, Q,, BEG, M.U,, VISWANATHAN, PJI. and ZAIDI, SH. Biochemical changes caused by asbestos dust in the lungs of rats. Scand. }. work environ. A health 1 (1975) 50--53. The contents of collagen, hexosamine, phospholipids, and cholesterol and the activities of acid and alkaline phosphatases, glutamic oxaloacetate transaminase, glutamic pyruvate transminase, aldolase, hexokinase, and lactic dehydrogenase were determined, in the lungs of rats 150 days after the intra. tracheal injection of amosite, anthophyllite, and chrysotile, Anthophyllite did not cause any significant change, while amosite and chxysotile caused significant increases in the contents of collagen and mucopolysaccharides. Lactic dehydrogenase and acid phosphatase activities were increased by all the dusts, while the other enzymes were not seriously affected. The biochemical significance of the findings in relation to asbestosis was discussed. Key words: experimental pneumoconiosis, asbestos, enzyme activity, biochemical changes, lungs. The biochemical aspects of experimental asbestosis are being studied in this labo ratory, especially in relation to the solu bility of the dusts under physiological conditions (8, 9, 13, 15). Lysosomal and mitochondrial enzymes have already been found among the targets of the toxic ef fect of asbestos (2, 14). It was of further interest to study the effect of asbestos dust in vivo on soluble enzymes and some tissue constituents of rat lungs, since this aspect has not received sufficient atten tion. The findings are reported below. MATERIAL AND METHODS Dusts Amosite, anthophyllite, and chrysotile dust samples with a fiber size below 30 /t were prepared according to the procedure 1 Industrial Toxicology Research Centre, Lucknow, India. Reprint request to: Dr. Qamar Rahman, Industrial Toxicology Research Centre, Post Box No. 80, Lucknow- 226001, India. described by Zaidi (17). The data for the fiber size distribution are recorded in table 1. Experimental production of asbestosis The dust was suspended in physiological saline to give a 0.5 */ (w/v) suspension and was autoclaved at a pressure of 15 lb for 20 minutes. For each dust 10 male albino rats, weighing from 150 to 200 g, from an I.T.R.C. animal colony were intratracheally injected with a single dose -of 1.0 ml of the sterilized suspension. The control animals received only saline. The animals were,maintained on a laboratory stock diet (18) 'and were sacrified by desanguination after 150 days. Estimation of chemical constituents For chemical analysis a weighed portion of lung was dried at 100 "C to a constant weight and powdered. Fifty milligrams of the dry powder was used for collagen estimation according to the method of -50 \ f Table 1. Percentage of different fiber lengths in the asbestos dust samples. Fiber length a Amosite Anthophyllite Chrysotile V. V* V. <3 3-- 5 6--12 13--17 18--24 25--29 21.3 30.0 30.3 6.1 7.9 4.3 23.3 34.9 25.7 6.6 5.5 4.1 13.4 15.7 20.6 13.9 16.8 19.5 vate transaminase (E.C. 2.6.1.2), alkaline phosphatase (E.C. 3.1.3.1), and add phos phatase (E.C. 3.1.3.2) were determined ac cording to Wootton (16). Total protein was determined, after preripitation with trichloroacetic add, according to Lowry et al. (7). RESULTS Chemical constituents Stegemaim (12) after extraction with ben zene. Phospholipids and free and bound cholesterol were estimated by the proce dures described by Wootton (16). Muco polysaccharides were estimated as glycosainine according to Ashwell (1). Enzyme assay Another weighed portion of lung tissue was cut into small pieces and homogenized in ice cold 0.25 M sucrose solution in a Potter-Elvehjem glass homogenizer. En zyme assays were carried out in the super natant after removing the mitochondrial fraction by differential centrifugation (10). Hexokinase (E.C. 2.7.1.1.), aldolase (E.C. 4.1.2.13), and lactic dehydrogenase (E.C. 1.1.1.27) were assayed by the procedures of Crane and Sols (4), Sibley and Lehninger (11), and Komberg (5), respectively. The activities of glutamic oxaloacetate transaminase (E.C. 2.6.1.1), glutamic pyru The data for the contents -of various chemical constituents in the asbestotic and control lungs are recorded in table 2. Amosite. Amosite caused an increase in fresh weight (83 /), dry solid content (44 */), and protein content (35 /). These changes were statistically significant (p < 0.1 /#, t-test). Collagen content per whole lung showed a 58 */ (p < 0.1 /) increase. Similarly expressed in terms of fresh weight, there was also a 41 */ increase. Hexosamine content indicated a 71 */ in1 crease in terms of fresh weight (p < 1 /#). Phospholipid, total cholesterol, and free cholesterol contents did not show any sig nificant change. Anthopkyllite. The fresh weight of lungs, dry solids, and protein did not show any change1 in the animals treated with anthophyllite as compared to the controls. Similarly the variations in col lagen, hexosamine, phospholipid, and cho lesterol were not statistically significant. Table 2. The mean the standard deviation of six determinations of the gross chemical changes in asbestotic rat lungs. Control Amosite Anthophyllite Chrysotile Animal weight* Fresh weight of whole lung b Dry solids b Protein b Collagen b Hexosamine b Phospholipid phosphorous b Total cholesterolb Free cholesterolb 180 25 1.27 0.28 182 10.5 99.2 10.5 10.9 1.14 1.16 0.52 0.77 0.04 7.25 0.93 5.16 0.72 175 24 2.33 0.68 '-242 *42 134 7.4 15.5 2.6 1.98 0223 0.66 0.09 9.31 0.12 5.29 0.18 a Expressed in grams b Expressed in milligrams per gram of fresh weight of lungs. 187 49 1.27 0253 204 9.7 97.8 3.8 12.5 1.4 1.41 0.41 0.70 0.09 6.30 0.11 5.14 1.0 172 35 3.42 1.5 228 31 135 3.4 14.9 1.6 2.18 0.17 0.70 0.04 7.61 0.37 4.66 0.37 51 ! Table 3. The mean the standard deviation of six determinations of enzymatic changes in the lungs of the asbestotlc and control rats. Enzymes a Control Amosite Anthophyllite Chrysotile Aldolase Hexokinase Lactic dehydrogenase Acid phosphatase Alkaline phosphatase Glutamic oxaloacetate transaminase Glutamic pyruvate transaminase 58.9 11.1 177 20 5JO 027 2.70 0.41 10.7 0.85 420 029 1.46 0.85 56.8 11.4 103 35 8.00 1.44 5.77 0.32 10.4 290 5.0 091 1.50 020 428 8.56 142 46 8.70 1.03 241 0.19 9.75 0.64 3.12 0.74 226 020 55.8 112 201 37 10.4 1.68 496 0.51 9.02 270 4.00 026 293 028 * Aldolase activity is expressed as [i moles of fructose 1, 6-diphosphate transformed, hexo kinase as u moles acid labile phosphate decreased, lactic dehydrogenase as u moles of reduced nicotinamide adenine dinucleotide oxidized, acid and alkaline phosphatase as milligrams of phenol liberated, and transaminases as.. moles pyruvate formed per gram of fresh tissue under the respective assay conditions. Chrysotile. The weight of the lung tis sue of the animals treated with chrysotile was 1.7-fold higher than that of the con trols. Dry solid content also registered an increase of 25 /. There was a 35 / in crease in protein content (p < 0.1 Vo). Col lagen content in milligrams per gram of fresh weight showed a 37 #/o increase. The intensity of collagen deposition became more apparent (2.6-fold increase) when the values were expressed per lung. Hexosamine content showed an 88 / in crease due to chrysotile, when expressed in terms of fresh weight (p < 0.1 Vo). Phospholipids, total cholesterol, and free cholesterol did not show any statistically significant change. Enzyme activities The activities of the various enzymes in the four groups of animals are recorded in table 3. Amosite. Fructose diphosphate aldolase activity did not show any change due to this dust. Lactic dehydrogenase and acid phosphatase activities were increased by 46 and 114 Vo, respectively (p < 0.1 /). Hexokinase activity tended to decrease by 42 Vo (p < 1 Vo). Alkaline phosphatase, glutamic pyruvate transaminase, and glutamic oxaloacetate transaminase were unaffected by amosite. Anthophyllite. The only significant change was a 58 % increase in lactic de hydrogenase (p < 0.1 Vo). Chrysotile. The activities of hexokinase, aldolase, glutamic oxaloacetate transami nase, and alkaline phosphatase showed no change. Lactic dehydrogenase and acid phosphatase increased by 69 and 84 Vo (p < 0.1 Vo), and glutamic pyruvate trans aminase by 100 Vo (p < 1 Vo). DISCUSSION The present results permit an understand ing of some of the chemical changes pro duced by asbestos in lungs, and they may be helpful therefore in understanding the biochemical basis of toxicity. Anthophyl lite did not produce any apparent chem ical changes in the present experiment, whereas the other two forms of asbestos caused significant alterations. This result is in agreement with the observation of differences in chemical composition, solu bility, and effect on mitochondrial en zymes (2, 8, 9) of this dust as compared to the other two. The increase in dry solid content in the asbestotlc lung indicated that relative water content varied between the control and experimental animals. Therefore, it was not considered as a suitable para meter for expressing the contents of var ious constituents for comparative pur poses. In terms of fresh weight collagen and hexosamine contents were found to accumulate in the lungs of animals treated with amosite and chrysotile. This finding agrees with the observation of reticulin type fibrosis in animals treated with amo- 52 i ; j tT( i1 I t 1 l <, l site (13). In addition to collagen noncol lagen protein (total protein-collagen) also tended to increase. This result may be in dicative of the role of extrapuimonary proteins in the process of asbestos body formation (3). The increase in lactic dehydrogenase, exhibited by all the three dusts, may be indicative of a metabolic adaptation to anaerobic conditions as reported for sili cosis (6) and may also be related to the role of macrophages in foreign body re action. The changes in hexokinase, aldcblase, alkaline phosphatase, glutamic oxaloacetate transaminase, and glutamic py ruvate transaminase were not significant enough to suggest metabolic alterations. Another interesting feature of the find ings is that asbestos dusts which inhibited transaminases and phosphatase activities of lung homogenate in vitro (9) showed a different effect in vivo. It may be point ed out that unlike the enzymes in the soluble fraction, membrane-bound en zymes of the mitochondria and lysosomes seem to be the more likely targets of as bestos toxicity in vivo (2, 14). Moreover, the increase in acid phosphatase observed in the case of animals treated with chrysotile and amosite is in agreement with the results on lysosomal enzymes (14). ACKNOWLEDGMENTS Thanks are due to Mr. M. M. Lai for supplying the dusts and Mr. S. D. Pandey for his technical assistance. REFERENCES 1. ASHwkJ.T,., G. Colorimetric analysis of sugars. In: S. P. COLOWICK and N. O. KAPLAN (eds.), Methods in enxymology (voL 3). Academic Press, New York, N.Y. 1957, pp. 73--105. 2. BEG, M. U., RAHMAN, Q,, VISWA NATHAN, P. N. and ZAIDI, S. H. The effect of asbestos dust on mitochondrial enzymes of rat lung. Environ- physiol. * biochem. 3 (1973) 185--191. 3. BLOUNT, M., HOLT, P. F. and LEACH, A. The protein coating of asbestos bodies. Biochem. j. 101 (1966) 204--207. 4. CRANE, R. K. and SOLS, A- Animal tissue hexokinases. In: S. P. COLOWICK and N. O. KAPLAN (eds.). Methods in enzymolopy (voL 1). Academic Press, New York, N.Y. 1955, pp. 277--286. 5. KORNBERG, A- Lactic dehydrogenases of muscle. In: S.P. Colowick and N. O. KAP LAN (eds.). Methods in enzymology (voL I). Academic Press, New York, N.Y. 1955, pp. 441--M3. 6. LINDY, S,, KAHANPAA, K, KARHTXNEN, P, HALME, J. and UITTO, J. Lac tate dehydrogenase isoenzymes during the development of experimental fibrosis. J. lab. clin. med. 76 (1970) 756--760. 7. LOWRY, O. HL, ROSEBROUGH, N. J, FARR, A. L. and RANDALL, R-J. Protein measurement with Folin phenol reagent. J. biol. chem. 193 (1951) 265--275. 8. RAHMAN. Q,, VISWANATHAN, P. N. and TANDON, S. K. In vitro dissolution of three varieties of asbestos in physiological fluids. Work-environ.-health II (1974) 39-- 42. 9. RAHMAN, Q., BEG, M. U, VISWA NATHAN, P. N. and ZAIDI, S. H. Relation between solubility of silicates and enzyme inhibition in lung homogenates (in vitro studies). Environ, physiol. <& biochem. 3 (1973) 281--285. 10. SCHNEIDER, W. C. Methods for the iso lation of particulate components of the cell. In: W. W. UMBREIT, R. H. BURRIS and J. F. STAUFFER (eds.). Monometric techniques. Burgess Publishing Co, Minne apolis, Minn. 1968, pp. 177--192. 11. SIBLEY, J. A. and LEHNINGER, A. L. Determination of aldolase In animal tissues. J. biol. chem. 177 (1949) 859--872. 12. STEGEMANN, H. Micro determination of hydroxyproline with chloromine-T and Pdimethyl aminobenzaldehyde. Hoppe Sel lers Z. physiol. Chem. 311 (1958) 41--45. 13. VISWANATHAN. P. N,, DOGRA, R- K. S, SHANKER, R. and ZAIDI, S. H. Pulmon ary fibrogenic response of guineaplgs to amosite dust. Jnt. Arch. Arbeitsmed. 31 (1973) 51--59. 14. VISWANATHAN, P. N, RAHMAN, Q, BEG, M. U. and ZAIDI, S. HL Pulmonary lysosomal enzymes in experimental asbestosis in guineapigs. Environ, physiol. & biochem. 3 (1973) 120--126. 15. VISWANATHAN, P. N, ANAND, ML, RAHMAN, Q, BEG, M. U. and ZAEDL S. H. Biochemical changes in serum of guineapigs in experimental asbestosis. Chemosphere 3 (1973) 119--124. 16. "WOOTTON, L D. P. Micro analysis m medical biochemistry. J. and A. Churchill Ltd, London 1964, pp. 83--86, 101--105, r- 112--114. 17. ZAIDI, S. H. Experimental methods. In: Experimental pneumoconiosis. Johns Hop kins Press, Baltimore, Md. 1969, pp. 35--19. 18. ZAIDI, S. H. and KAW, J. D. Effect of dietary deficiency and protein malnutrition on the librogenesis caused by silica dusts in rats. Br. j. ind. med. 27 (1970) 250--259. Received for publication: 1974-06-12 J 53 Scand. j. work environ. & health X (X975) 54--59 Blood cell d -aminolevulinic acid dehydratase activity in humans exposed to methylmercury by ANDREJS SCHt)TZ, B.Sc.,1 and STAFFAN SKERFVTNG, M.D.1- - SCHOTZ, A. and SKERFVING, S. Blood cell d-aminolevulinic acid dehydratase activity in humans exposed to methylmercury. Scand. j. work environ. & health 1 (1975) 54--59. The (^aminolevulinic acid dehydratase (ALA-D) activity in blood cells was studied in 15 subjects exposed to methylmercury through consumption of con taminated fish and 19 <unexposed subjects with a similar sex and age distribu tion. The exposed subjects had a mean mercury level of 120 (range 15--370) ng/g blood cells while the controls had 9 (range 4--15) ng/g. Both groups bad the same mean level of lead in whole blood (101 g/100 ml). ALA-D activity decreased statistically significantly as both mercury and lead levels in the blood cells increased. Key words: toxicology, human experiment, d-aminolevulinic acid dehydratase, methylmercury, lead, blood. Methylmercury in fish has grown into a major toxicological concern in many coun tries (3, 10, 20). Interest has so far been focused mainly on nervous tissue damage (2). It would be of great importance to know also if effects occur at lower de grees of exposure. In the case of inorganic lead even a minor increase in the blood lead level causes a significant decrease in the activ ity of the blood cell <5-aminolevulinic acid dehydratase (ALA-D; E.C. 4.2.1.24) (5, 15, 16, 17, 18), an enzyme engaged in the heme synthesis. The sulfhydryl groups in ALA-D are necessary for enzymatic acti vity (14). Both inorganic lead and methyl mercury bind to sulfhydryl groups in pro teins and accumulate in the blood cells. The addition of inorganic mercury salt to hemolyzed human blood causes a decrease of ALA-D activity (7). Thus it was of in terest to study whether ALA-D activity was depressed in subjects exposed to methylmercury through fish consumption. 1 Department of Occupational Medicine, Uni versity Hospital, Lund, Sweden. - Research Department, National Food Ad ministration, Stockholm, Sweden. Reprint requests to: Mr. Andrejs Schutz, De partment of Occupational Medicine, University Hospital, S-221 85 Lund, Sweden. MATERIAL AND METHODS Subjects The exposed group contained 15 subjects (12 males aged 44--76 years and 3 females aged 53--56), who consumed various amounts of fish from four mercury-con taminated water areas in central Sweden. The average methylmercury levels in pike (Esox lucius) from those areas ranged from 0.5 to 6 mg of mercury per kilo gram of wet weight. As reported in an earlier study no one had symptoms or signs of methylmercury poisoning (30). ` Blood samples were also obtained from 19 unexposed* subjects from urban areas of Stockholm- and Lund. Their sex and age distribution was very similar to the exposed group. .They had fish once a week or less. None of the unexposed had eaten fish from mercury-contaminated water areas. None of the exposed or unexposed sub jects had a history of occupational expo sure to mercury or lead or of exposure to mercury-containing drugs. None had a record of alcohol abuse, and no one had consumed alcohol close to sampling. 54