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J 352 hAl UML VUL d+j u * Wildenthal K. J.. Fmr. rtn.inl. .W.. 207. Jt (I7r.7). Wiidcniii;ii k. I..J. Arri riitu-i.. Ji. I5J fi77n. * WiUlcinlul. K. J: I'/mw../, .s.ir.. 217. (1771). * Wildenthal. K. 3.. Amrr.J. /Viu>,i).. 221. 'IK (1771). * Hughes, 13. M,, and Lnnemnic. I). It.. Aw/ine. 235. J.U (1772). * Lnngmore, D. H.. and Hughes, I). M.. Sniurr, 2.IK, JO (1972). * Naglcr, J., and Longmore, D. U., Avrurr. 242, 197 (I97J). Days Fig.1 Ventrieulir rites or the control hearts compared with the first 23 d for the test hearts. seem reasonable to continue with further conventional clinical trials, if a new drug is depressant to the foetal myocardium above a certain concentration, it would be unwise to exceed this in a subsequent clinical trial. If the substance were to affect only young foetal hearts it might still be -:fe in late pregnancy. We thank the Board of Governors of the National Heart and Chest Hospitals and the British Heart Foundation W financial support, Miss D. M. Hughes and our colleagues --r help and Dame Honor Fell and Kern Wildenthal for encc jra-ement. National Heart Hospital, S. R.'Ahmithono D. B. Westmoreland Street, London H'lM BSD Received January 23; revised April 10.1973. v Penetration of Asbestos through the Digestive Tract of Rats MESOniruoMAS of the pleura and peritoneum1 "* have been linked with the inhalation of asbestos fibres. We have shown* that asbestos fibres arc present in drinking water, beer, wine and other beverages. Asbestos fibres can penetrate the mucosa of the stomach and intestine1 and Tclischi and Rubenstone* have found asbestos material in a gastric carcinoma. Godwin and Jagatie1, reporting on mesotheliomas of the pleura caused by asbestos, noted that asbestos particles were widely distri buted in various tissues. They suggested that panicles may migrate from the lung through the blood and lymphatic system to all parts of the body. In the light of these repons, we surmised that asbestos consumed orally can pass through . the gut into the blood stream and accumulate in various tissues. To determine if this is the case we injected a suspension of asbestos fibres into the stomachs of rats. The suspension was prepared by shaking 2.5 g of chrysotile asbestos CJohns Manville No. 7RF02) in 300 ml of water in a graduated cylinder. The suspension was allowed to settle for 30 min and the top 230 ml, which contained about 9.4 x 10' (1 mg) fibres ml*1, was drawn off. Most of these fibres are 0.2 pm2.0 pm long (Fig. 1). To ensure that fibres passed through the digestive tract without rats inhaling any of them, the asbestos was introduced into the gastrointestinal tract by opening the abdomen under anaesthesia and injecting the fibres directly into the stomach. Two to 4 d later the rats were killed and blood and tissues analysed for asbestos. To ensure that any fibres on the rat hair could not contaminate the organs removed during surgery, the rats wrere first thor oughly vacuumed and washed with double distilled water and methanol. During the removal of tissues wet towels were placed over the animals and around the incisions. Uncon taminated blood was drawn from the orbital sinus using a capillary tube. To avoid formation of excess ash, which interferes with examination of specimens under the electron microscope, the " tissues were first solubili2ed with soluene (Packard Instrument Company): the fibres were then centrifuged down, washed with methanol and ashed as previousiv described*. The ash was taken up in I ml of distilled water hich had been filtered through a 0.2 pm filter, and 3 pi of this mixture was dropped on hydrophilic carbon-coated electron microscope grids supported on a ring peg. The grids were dried in a closed glass container (to avoid air contamination) under a heat lamp and were examined at magnifications of 20,000 and 80,000 with an electron microscope. The presence of ch'rysotilc asbestos fibres was verified by electron dillrncnon. They were counted and the numbers corrected fur dilution to determine the number in the original I ml sample. Results of recovery of asbestos fibres from rats treated in this way are shown in Table I. Rats in group I were given 9.4x 10* fibres (I ml) and killed 2 d later; those in group 2 received 94 * 10* fibres and w-ere killed 4 d later. No asbestos fibres were detected in the blood of non-injccted rats (controls) but 4.63 x 10* g*1 (a statistically significant increase) were found in the first group. This is the first time direct physical evidence for the presence of asbestos fibres in the blood has been obtained. Four days after treatment the amount of asbestos in the blood of rats in group 2 had dropped to 1.19* 10* g*` even though these rats received ten times as much asbestos. This indicates that b'nod NATURE VOL 243 JUNE 8 1973 353 0 Table t Numbatt ol Asbestos Ftb'ts Raeovatad.ltom Ttaaud and ' Non-Tfaatad Ran ' Blood Spleen Omenrum Heart Brain Lungs Controls mean* 0.00 2.33 0.67| 2.46 0.46 2.09 0.41 0.05 0.02 1.06 0.26 Group 11 mean 4.6511 4.11 2_.74 1.02 0.49 0_.87 0-313 0.19 1.10 034 Group mean 2! 1.19 3.43 1835 238 039* 1.74 0.77 130 7.79 0.40 0.11 036 * Avrrxpe of five rati in all cam except where noted, f Animab injected with 9.4 x 10* fibres and liuuei examined 2 d later. . t Antnull injected with 94 x 10* fibres and tissues examined 4 d later. {Standard error of the mean. 8 Four rats only. ^ 7*<0.01: P<0.0i. (The figures in the table arc fibres g~` x 10-4.) can clear itself of the fibres. Tissues such as the spleen, heart and lung also seem to have, to a lesser extent, the ability to clear themselves of the asbestos fibres, as the figures obtained from rats in group 2 indicate. They are lower than those for group 1 rats even though they received a larger dose, but the tissues had longer to clear themselves. Indeed, recent work in our laboratory with radioactive asbestos indicated that some tissues, such as the lungs, can reduce their fibre content by half in 2 d. This clearing action could possibly have quite an effect on the numbers of fibres found in the liver and kidney of the rats but unfortunately, because of the considerable amount of ash present in these organs even after soluene treatment, we could not examine these specimens under the electron microscope. Another factor niay contribute to the higher fibre-count obtained for the organs of rats in group I compared with animals in group 2, namely, that there were four times as many fibres in the blood of rats in group I. The residual blood in the tissues on removal from the animal would of course contribute lo the total fibre count of a particular tissue. This factor could have created the anomaly in the fibre counts in brains. In the brains of treated rats this was about six times higher than in the controls, and the increase in fibre count is statistically significant for rats in group 2 but not, oddly, for rats in group 1 even though there were more fibres in these animals. The greater number of fibres per gram in the blood of rats in group 1 could be responsible for the larger standard error in the counts of rats in group 1 and therefore did not allow the values obtained to be significant. The brain seems to have little ability to clear itself of asbestos fibres, but the omentum has even less. This tissue, which /- '* rr ~,'r " L. .. . I/ms Fig. 2 Small bundle of asbestos fibres from the peritoneum of a rat. This bundle is at least 13 pm long. surrounds the small intestine, appeared to accumulate most ' asbestos. Although the asbestos fibre content of the omentums in rats in group 2 may seem rather high (one extreme count contributed to the high standard error) the omentum does not seem to respond as do the other tissues. As well as having little ability to clear itself, fibre accumulation in the omentum may take place much more slowly than it does in other tissues. As the omentum consists partly of peritoneum and partly adipose tissue, asbestos fibres may enter the peritoneum by - direct penetration of the intestinal wall. Concerning the prolonged retention of asbestos fibres in the omentum in women asbestos workers, Keal* found a higher incidence of peritoneal cancer than of lung cancer. The amount of asbestos found in the tissues of the controls is higher than we had anticipated, considering the age of the rats and the number of fibres they might have consumed in their drinking water*. But wc do net know how much was in their feed or air. We have analysed the tissues of some people who died of natural causes and found somewhat similar levels. In one person there was twice as much asbestos in the brain as in the brains of our control rats and about one quarter - as much in the omentum, but the omentum content was still higher than the brain. Most of the fibres found were about 0.2-2.0 pm long but tome measured 13 pm (Fig. 2) and one fibre in the blood of a rat was 23.55 pm long. We do not know how the fibres pass through the intestinal wall. Some long ones may pierce the gut like a needle, whereas pinocytosis may Account for the absorption of the small ones. We demonstrated earlier Ihe presence of asbestos fibres in air, snow, city drinking water and a number of beverages*. The experiment described here shows that fibres of a similar size administered into the stomach of mis appear in the blood and accumulate in various tissues in the body. R. D. PovtcpkaCT H. M. Cunningham Food Research Laboratories, Health Protection Branch. Department of Nationo/ Health and Welfare, Ottawa | - * ; lie. I flretron miffnyrjph of the suspension or asbestos fthTi injeeird imo the rat stomach. Note the \-arying suet of t ic bundles of fibres present. Received November 30, 1973; revised February 13. 1973. * Hourihane. D. O. n.. Thera r. 19. 261 (19651 * Hourihane. D. O. U.. Ann. NY Arod. Sri.. 131. M7 (1965). * Lynch. K. M.. and Smith. \V. A_ Amer.J. Cenrrr.U. 56 119351. * Cunningham. II. M.. and f'onicfract, R. D,, Aeture, 2J2. 332 f 19711. * SelilofT. I. L. Chnrg. and Hammond, E. C.. J. Amrr. Mi d. A***W: fkk. 674 (IW.J)' * Telitchi. M.. and l(titvn>iniir. A. I.. Arch. Potho!., 72 (1961). * Godwin. M. C.. and lajouic. }.. Lauren. A\i,, 3, 391 (1970). ' Keal. E. E-. Lanret, ii, |;u (|V60l 0 i 2 i * i b i ionwuN|)ii,i;ix ivji:i 222.124 b x. . teem reasonable lo continue with further conventional clinical trials. If a new drug is depressant to the foetal myocardium above a certain concentration, it would be unwise to exceed this in a subsequent clinical trial. If the substance were to alTect only young foetal hearts it might still be afe in late pregnancy. We thank the Board of Governors of the National Heart and Chest Hospitals and the British Heart Foundation r?r financial support, Miss D. M. Hughes and our colleagues : ~.r help and Dame Honor Fell and Kern Wildenthal for enccora-emcnt. S. R. 'A kmstrono D. B. Lo ^omori National Heart Hospital, Westmoreland Street, London H'lM BSD Received January 25; revised April 10.1973. ` HAlUMl t'OL. vwmi v Wildenthal K. J.. JVw. /Vm .W.. 207. 33 f lO/.Oj. * Wildenthal! K. J.. A Appl /V.i >,,!.. .HI. 153 119711. . * XVihlenllul, K. J.. h"1'' /7#i uiZ. .\.*r.. 717. 5t (1771). * Wildenthal. K. J.. Anter, J. Plnuitl.. 271. I.<H (1771). * llughvs, |J. M,, and Unrmrac. I). It.. Aniwti*. 235. 334 (1972). * Longmnre, D. It., ami lliipht't. I). M.. Ainure, 23K, 40(1972). * Naglcr, J,, and Longmont, O. tt.. Suture, 242, 197 (I97J). v Penetration of Asbestos through the Digestive Tract of Rats Mesotsicuomas of the pleura and peritoneum'-* have been linked with the inhalation of asbestos fibres. We have shown* that asbestos fibres arc present in drinking water, beer, wine and other beverages. Asbestos fibres can penetrate the mucosa of the stomach and intestine' and Telisehi and Rubenstone* have found asbestos material in a gastric carcinoma. Godwin and Jagatic*, reporting on mesotheliomas of the pleura caused by asbestos, noted that asbestos particles were widely distri buted in various tissues. They suggested that panicles may migrate from the lung through the blood and lymphatic system to all pans of the body. In the light of these repons, we surmised that asbestos consumed orally can pass through . the gut into the blood stream and accumulate in various tissues. To determine if this is the case we injected a suspension of asbestos fibres into the stomachs of rats. The suspension was prepared by shaking Z5 g of chrysolite asbestos (Johns Manville No. 7RF02) in 500 ml of water in a graduated cylinder. The suspension was allowed to settle for 30 min and the top 250 ml, which contained about 9.4 x 10* (1 mg) fibres ml"', was drawn off. Most of these fibres are 0.2 pm2.0 pm long (Fig. I). To ensure that fibres passed through the digestive tract without rats inhaling any of them, the asbestos was introduced into the gastrointestinal tract by opening the abdomen under anaesthesia and injecting the fibres directly into the stomach. Two to 4 d later the rats were killed and blood and tissues analysed for asbestos. To ensure that any fibres on the rat hair could not contaminate the organs removed during surgery, the rats were first thor oughly vacuumed and washed with double distilled water and methanol. During the removal of tissues wet towels were placed over the animals and around the incisions. Uneontaminated blood was drawn from the orbital sinus using a capillary tube. To avoid formation or excess ash, which interferes with examination of specimens under the electron microscope, the " tissues were first solubilized wiih soluene (Packard Instrument Company); the fibres were then centrifuged down, washed with methanol and ashed as previously described*. The ash was taken up in I ml of distilled water *hich had been filtered through a 0.2 pm filler, and 5 pi of this mixture was dropped on hydrophilic carbon-coated electron microscope grids supported on a ring peg. The grids were dried in a closed glass container (to avoid air contamination) under a heat lamp and were examined at magnifications of 20.000 and 80,000 with an electron microscope. The presence of ch'rysotile asbestos fibres was verified by electron dillraciion. They were counted and the numbers corrected fur dilution to determine the number in the original I nil sample. Results of recovery of asbestos fibres from rats treated in this way are shown in Table I. Rats in group I were given 9.4 x 10* fibres (I ml) and killed 2 d later; those in group 2 received 94 x 10* fibres and were killed 4 d later. No asbestos fibres were detected in the blood of non-injectcd rats (controls) but 4.65 x 10* g~* (a statistically significant increase) were found in the first group. This is the first time direct physical evidence for the presence of asbestos fibres in the blood has been obtained. Four days after treatment the amount of asbestos in the blood of rats in group 2 had dropped to I.J9x 10* g'1 even though these rats received ten times as much asbestos. This indicates that b'aod *. N0 ATURE VOL 243 JUNE 8 1973 353 'Ttbl* 1 Number* of Aibeslo* Fibre* Recovered. Irom Treated end - Non-Treited Rate ' Blood Spleen Omentum Heart Brain Lunp Controls mesa* 0.00 2.33 0.67| 2.46 0.46 2.09 0.41 0.03 0.02 1.06 026 Group 11 mean 4.631 1.02 4.11 0.49 2.74 0*7 OJIJ - 0.19 1.10 0.54 Group 21 mean* 1.19 J.43 11.25 228 0.29** 0.77 120 7.79 0.40 0.11 1.74 026 * Aver*pc of five rati in all airs except where noted, f Animals ipjeeted with 9.4 x 10* fibres and tissues examined 2 d later. . t Ani mats injected with 94 x 10* fibres and tissues examined 4 d later. (Standard error of the man. (Four rats only. 1 7*<0.01; R<0.03. (The figures in the table are fibres fx 10**.) can clear itself of the fibres. Tissues such as the spleen, heart L. .. . 'ITM and lung also seem to hate, to a lesser extent, the ability lo clear themselves of the asbestos fibres, as the figures obtained Fig. 2 Small bundle ofasbestos fibres from the peritoneum of a rat. This bundle is at least 13 pm long. from rats in group 2 indicate. They are lower than those for group ) rats even though they received a larger dose, but the tissues had longer to clear themselves. Indeed, recent work in our laboratory with radioactive asbestos indicated that some tissues, such as the lungs, can reduce their fibre content by half in 2 d. This clearing action could possibly have quite an effect on the numbers or fibres found in the liver and kidney of the rats but unfortunately, because of the considerable amount of ash present in these organs even after soluene treatment, we could not examine these specimens under the electron microscope. Another factor may contribute to the higher fibre-count obtained for the organs of rats in group I compared with animals in group 2. namely, that there were four times as many fibres in the blood of rats in group I. The residual blood in the tissues on removal from the animal would of course contribute to the total fibre count of a particular tissue. This factor could have created the anomaly in the fibre counts in brains. In the brains of treated rats this was about six times higher than in the controls, and the increase in fibre count is statistically significant for rats in group 2 but not, oddly, for rats in group 1 even though there were more fibres in these animals. The greater number of fibres per gram in the blood of rats in group I could be responsible for the larger standard error in the counts of rats in group ) and therefore did not allow the values obtained to be significant. The brain seems to have little ability to dear itself of asbestos fibres, but the omentum has even less. This tissue, which surrounds the small intestine, appeared to accumulate most asbestos. Although the asbestos fibre content of the omentums in rats in group 2 may seem rather high fonc extreme count contributed to the high standard error) the omentum does not seem to respond as do the other tissues. As well as having little ability lo dear itself, fibre accumulation in the omentum' may lake place much more slowly than it does in other tissues. As the omentum consists partly of peritoneum and partly adipose tissue, asbestos fibres may enter the peritoneum by direct penetration of the intestinal wall. Concerning the prolonged retention of asbestos fibres in the omentum in women asbestos workers, Keal* found a higher incidence of peritoneal cancer than of lung cancer. The amount of asbestos found in the tissues of the controls is higher than we had anticipated, considering the age of the rats and the number of fibres they might have consumed in their drinking water4. But we do net know how much was in their feed or air. We have analysed the tissues of some people who died of natural causes and found somewhat similar levels. In one person there was twice as much asbestos in the brain as in Ihe brains of our control rats and about one quarter . as much in the omentum, but the omentum content was still higher than the brain. Most of the fibres found were about 0.2-2.0 pm long but tome measured 13 pm (Fig. 2) and one fibre in the blood of a rat was 23.S5 pm long. We do not know- how the fibres pass through the intestinal wall. Some lone ones may pierce the gut like a needle, w-hereas pinocytosis may account for the <-r'* n*i*f -- absorption of the small ones. We demonstrated earlier the presence of asbestos fibres in air, snow, city drinking water and a number of beverages4. The experiment described here shows that fibres of a simitar size administered into the stomach of rats arpear in the blood and accumulate in various tissues in the body. R. D. PovrrrxACT H. M. Cunningham Food Research Laboratories, Health Protection Branch. Department of national Health and Welfare, Ottawa i L -- U-V" wfe W % 4M Hr. I Mrciron mirrnjruph of the suspension of asbestos fibers injected into the rat siomaeh. Note the varying sues of i :e bundles of fibres present. Received November 30, 1972; revised February 13. 1973. Hourihane. 13. O. ft.. Thoras. 19. 263 (19631 * Hourihane. D. O. II.. Ann. a'Y Arad. Sri.. 132. M7 (1963). * Lynch. K. M,, and Smith. W. A_ Aimr. J. Cancer. 24. 36 f 1935). 4 Cunningham. II. M.. and I'oniefract, R. D.. nature. 132. 332 (1971). * Selikofl. I. Chnrg. and llammond. E. C.. J. Amrr. hUd. Atvtr.. IK*. 674 (I`JMI. * Tebschi. M.. and l(iihvn,lmie. A. I.. Arrh. Pathol.. 72 (1961). * Godwin, M.C.,amJ lapuie,!.. Lnuran. A,-j, 3, 391 (1970). 4 Keal. E. C.. Lmret, ji, |;|| (|60)