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NATURE VOL. 243 JUNE 8 1973 Table 1 Summary of Effects of Test Drugs Drug (pg ml- ') Atria Ventricles Control Exponential decay. Exponential decay. Beating rate slower than atria and ceases sooner. Tyramine 40 Overall depression of Similar to controls. beating rate. Decrease in survival time. Oxyfedrine 20 The curve swung to a more horizontal posi tion. 0-8 d beating slowed. 9-19 d rate speeded up. Slight in crease in survival time. Curve swung to more horizontal position. Rate decreased up to day 4, rate increased from day 5-16. Sur vival time increased, Oxyfedrine 40 Slight but uniform in crease in rate from day 2 to day 15. No in crease in survival time. Very slight decrease in rate up to day 6, in crease in rate from day 7 to 14. No in crease in survival time. Oxyfedrine) Tyramine J Similar to control; the Similar to control. two drugs cancelling Drugs cancel. each other out. Oxyfedrine 80 Similar to control; Uniform slight increase striking reduction in in rate. Reduction in the variability o f the scatter as for atria. rate. (Scatter.) Isoprnaline 50 Moderate overall in Moderate overall in crease in rate but no crease in rate; no increase in survival increase in survival. time. Greater scatter. L-Noradrenaline 15 Similar to oxyfedrine 20. Curve swung to horizontal position. Decreased rate 0-8 d, increased rate days 9 23. Survival time in creased more than by oxyfedrine 20. No significant effect from day 0-5; in creased rate from day 6-17. Increase in sur vival time. Practolol 50 0-8 d similar control, 9-18 d rate increased. Slight increase in sur vival time. Overall increase in rate starting slight and be coming large. Large increase in survival time. 351 d The four hearts under each set o f conditions represent a range o f foetal ages, and therefore a range o f developmental stages, giving a certain amount o f variation in beating perform ance. The performance o f the youngest and oldest groups o f hearts were examined separately, and predictably the youngest hearts lived longer and beat better than the oldest. The atrial and ventricular beating rates o f the oldest control hearts were erratic in this preliminary series, and we suggest that for drug evaluation, hearts o f less than 60% o f term give more meaning ful results. Som e interesting differential effects were demonstrated. Oxyfedrine, for example, at 20 pg m l'1 showed a positive effect on both atria and ventricles in the young hearts with no apparent effect on the beating o f the older atria and ventricles ; whereas isoprnaline affected both the young and old hearts equally. As well as being a potentially useful assay for cardioactive drugs, it seems also that any substance which is to be given to pregnant women should be examined for its direct effect on the m ouse foetal heart. Further study with human foetal heart should follow if any effect is demonstrated. It cannot be assumed, however, that the substance is harmless if there is no effect on mouse or human foetal heart; many drugs are not pharmacologically active in their pure form, whereas their breakdown products are. Complete drug evaluation will require cultures with each o f the intermediate breakdown products at their likely concentrations. If after this screening a drug seems to have no effect, or even a beneficial one, it would Days Fig. 1 Atrial rates o f the control hearts compared with the atrial rates o f the test hearts for the first 23 d o f culture. Hearts from nine litter mates o f comparable size were selected from pregnant mice for each set of experiments. (When one pregnant mouse contained only seven foetuses, two foetuses of the same crown rump size from another litter had to be used.) The experiment was repeated successfully four times, using a range of foetal ages 54% to 80% term (crown rump 9 to 16 mm). Hearts were cultured as previously described5 on stainless steel mesh so that their lower surfaces were wetted by 65 % medium 199 (Burroughs Wellcome), with antibiotics, 35% foetal calf serum, 50 pg m l-' insulin, 0.1 pg m l-' cortisol; in a water vapour saturated atmosphere o f 95 % oxygen and 5 % C 0 2 and at a temperature o f 37 C 2 C. Six drugs were added to this standard medium to give the nine test media, as follows: (a) standard medium (control); (6) medium-)-tyramine hydro chloride 40 pg ml-1 ; (c) medium +oxyfedrine 20 pg m b 1; (d) medium-)- oxyfedrine 40 pg ml"1; (e) medium-)- tyramine hydro chloride 40 pg ml-1 plus oxyfedrine 40 pg ml"1; ( / ) medium + oxyfedrine 80 pg ml-1 ; (g) medium + isoprenaline 50 pg ml"1; (h) medium + L-noradrenaline 15 pgm l"1; (i) medium+ practo lol 50 pg ml-1 . The medium was replaced every other day after observations had been made. The cultures were examined daily using a dissecting microscope, during which examination special efforts were made to maintain the same constant conditions of temperature, atmosphere and water vapour saturation. 352 l 2 3 4 5 6 7 8 9 IO U I 2 l 3 l 4 | 5 i 6 l 7 l 8 I920 21 22 23 24 b "V - ,. g h 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 safe in late pregnancy. We thank the Board of Governors o f the National Heart and Chest Hospitals and the British Heart Foundation for financial support, M iss D . M. Hughes and our colleagues for help and Dam e H onor Fell and Kern Wildenthal for encouragement. S. R. A rmstrong D . B. Longmore National Heart Hospital, Westmoreland Street, London W \M 8B D Received January 25; revised April 10,1973. NATURE VOL. 243 JUNE 8 1973 1 Wildenthal, K. J., Proc. Physiol. Soc., 207, 33 (1969). 2 Wildenthal, K. J., J. Appl. Physiol., 30, 153 (1971). 3 Wildenthal, K. J., Proc. Physiol. Soc., 217, 56 (1971). 4 Wildenthal, K. J., Amer. J. Physiol., 221, 238 (1971). 5 Hughes, D. M., and Longmore, D. B., Nature, 235, 334 (1972). 6 Longmore, D. B., and Hughes, D. M., Nature, 238, 40 (1972). 7 Nagler, J., and Longmore, D. B., Nature, 242, 197 (1973). Penetration o f Asbestos through the Digestive Tract o f Rats M esotheliomas o f the pleura and peritoneum 1-3 have been linked with the inhalation o f asbestos fibres. W e have shown4 that asbestos fibres are present in drinking water, beer, wine and other beverages. Asbestos fibres can penetrate the m ucosa o f the stomach and intestine5 and Telischi and Rubenstone6 have found asbestos material in a gastric carcinoma. Godwin and Jagatic7, reporting on mesotheliomas o f the pleura caused by asbestos, noted that asbestos particles were widely distri buted in various tissues. They suggested that particles may migrate from the lung through the blood and lymphatic system to all parts of the body. In the light o f these reports, 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 o f rats. The suspension was prepared by shaking 2.5 g o f chrysotile asbestos (Johns Manville N o. 7R F 02) in 500 ml o f 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 109 (1 mg) fibres m l-1 , was drawn oif. M ost o f these fibres are 0.2 p m 2.0 pm long (Fig. 1). T o 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. Tw o 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 o f tissues wet towels were placed over the animals and around the incisions. U ncon taminated blood was drawn from the orbital sinus using a capillary tube. To avoid formation o f excess ash, which interferes with examination o f specimens under the electron microscope, the tissues were first solubilized with soluene (Packard Instrument Company); the fibres were then centrifuged down, washed with methanol and ashed as previously described4. The ash was taken up in 1 m l o f distilled water which had been filtered through a 0.2 pm filter, and 5 pi o f this mixture was dropped on hydrophilic carbon-coated electron m icroscope 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 o f chrysotile asbestos fibres was verified by electron diffraction. They were counted and the numbers corrected for dilution to determine the number in the original 1 ml sample. Results o f recovery o f asbestos fibres from rats treated in this w ay are shown in Table 1. Rats in group 1 were given 9.4 x 109 fibres (1 ml) and killed 2 d later; those in group 2 received 94 x 109 fibres and were killed 4 d later. N o asbestos fibres were detected in the blood o f non-injected rats (controls) but 4.65 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 o f asbestos fibres in the blood has been obtained. Four days after treatment the am ount o f asbestos in the blood o f rats in group 2 had dropped to 1.19 x 10 g -1 even though these rats received ten times as much asbestos. This indicates that blood NATURE VOL. 243 JUNE 8 1973 353 T able 1 Numbers o f Asbestos Fibres Recovered from Treated and Non-Treated Rats Blood Spleen Omentum 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 1 1 mean * 4.651J 4.11 2.74 -- 1.02 0.49 0.87 -- 0.3111 0.19 1.80 0.54 Group 2{ mean * 1.19 3.45 18.25 2.28 0.29** 1.74 0.77 1.20 7.79 0.40 0.11 0.26 * Average of five rats in all cases except where noted, t Animals injected with 9.4 x 10g fibres and tissues examined 2 d later, J Ani mals injected with 9 4 x l0 9 fibres and tissues examined 4 d later. Standard error of the mean. )| Four rats only. If P cO .O l; ** P < 0.05. (The figures in the table are fibres g_1 x 10-6 .) 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 o f 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 som e 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 o f fibres found in the liver and kidney o f the rats but unfortunately, because o f the considerable amount o f 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 o f rats in group 1 compared with animals in group 2, namely, that there were four times as m any fibres in the blood o f rats in group 1. The residual blood in the tissues on removal from the animal would of course contribute to the total fibre count o f a particular tissue. This factor could have created the anom aly in the fibre counts in brains. In the brains o f 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 o f fibres per gram in the blood o f rats in group 1 could be responsible for the larger standard error in the counts o f 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 o f asbestos fibres, but the omentum has even less. This tissue, which Fig. 1 Electron micrograph of the suspension of asbestos fibres injected into the rat stomach. N ote the varying sizes of the bundles o f fibres present. Fig. 2 Small bundle o f asbestos fibres from the peritoneum of a rat. This bundle is at least 15 pm long. surrounds the small intestine, appeared to accumulate most asbestos. A lthough the asbestos fibre content o f 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 o f peritoneum and partly adipose tissue, asbestos fibres may enter the peritoneum by direct penetration of the intestinal wall. Concerning the prolonged retention o f asbestos fibres in the omentum in women asbestos workers, K eal8 found a higher incidence o f peritoneal cancer than o f lung cancer. The am ount o f asbestos found in the tissues o f the controls is higher than we had anticipated, considering the age o f the rats and the number o f fibres they might have consum ed in their drinking water4. But we do not know how much was in their feed or air. We have analysed the tissues o f some people w ho died o f natural causes and found somewhat similar levels. In one person there was twice as much asbestos in the brain as in the brains o f our control rats and about one quarter as much in the omentum, but the omentum content was still higher than the brain. Most o f the fibres found were about 0.2-2.0 pm long but som e measured 15 pm (Fig. 2) and one fibre in the blood o f a rat was 23.55 pm long. W e do n ot 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 the presence o f asbestos fibres in air, snow, city drinking water and a number o f beverages4. The experiment described here shows that fibres o f a similar size administered into the stomach o f rats appear in the blood and accumulate in various tissues in the body. R. D . Pontefract H. M. Cunningham Food Research Laboratories, Health Protection Branch, Department o f National Health and Welfare, O ttaw a Received November 30, 1972; revised February 13, 1973. 1 Hourihane, D. O. B., Thorax, 19, 268 (1965). 2 Hourihane, D. O. B., Ann. N Y Acad. Sci., 132, 647 (1965). 3 Lynch, K. M,, and Smith, W. A., Amer. J. Cancer, 24, 56 (1935). 4 Cunningham, H. M., and Pontefract, R. D., Nature, 232, 332 (1971). 5 Selikoff, I. J., Churg, J., and Hammond, E. C., / . Amer. Med. Assoc., 188, 674 (1964). 6 Telischi, M., and Rubenstone, A. I., Arch. Pathol, 72 (1961). 7 Godwin, M. C., and Jagatic, J., Environ. Res., 3, 391 (1970). 8 Keal, E. E., Lancet, ii, 1211 (1960). 354 Presynaptic and Postsynaptic Effects o f Lead at the Frog Neuromuscular Junction L ead has long been known to be toxic to animals and humans, with som e o f its effects being attributable to actions on the neuromuscular system1*, Since several other polyvalent cations have potent effects on neuromuscular transmission3^19, the question arose as to whether one o f the manifestations o f lead toxicity might be an interference with neuromuscular trans mission. Answers to this question might elucidate some mechan isms o f lead toxicity and, at the same time, improve our under standing o f junctional transmission. This report shows that lead influences both pre and postsynaptic events in neuro muscular transmission with the presynaptic ones being most sensitive. There have been no previous electrophysiological studies o f the effects o f lead on neuromuscular transmission. However, K ostial and V ouk20, in a study o f the perfused superior cervical ganglion o f the cat, found that lead nitrate, in concentrations as low as 12.1 ijM , blocked ganglionic transmission. The authors concluded that this effect was due to a presynaptic action of lead because: (a) the acetylcholine (ACh) output o f the eserinized ganglion, resulting from preganglionic nerve stimula tion, was reduced by lead, and (>) the postganglionic response to perfused ACh was not affected by lead. Experiments were performed in vitro on the isolated sciatic nerve-sartorius muscle preparation of the frog (Rana pipiens). Superficial neuromuscular junctions were located optically with a compound microscope (magnification x 400). Standard microelectrode and photographic techniques were used to monitor intracellular responses from individual end-plates. R esponses were recorded first while the preparation was bathed in a control Ringer solution, then in the presence o f lead, added as PbClj, and finally after the lead had been washed out. Solutions entered the experimental chamber by gravity and left by suction. The composition o f the normal Ringer solution w as: 111 mM NaCI, 2.5 m M KC1, 2.0 raM C aC l2, 4 mM Tris-maleate. All Ringer solutions were maintained at pH 6.9 and at a temperature o f 15 C. Preparations were exposed to lead by bathing them in a Ringer solution to which 0.01-0.10 mM PbCla had been added. To record subthreshold end-plate potentials, nerve-muscle preparations were bathed in 0.8 mM C a2+/5.5 m M M g2+-Ringer solution3'21. A cetylcholine was applied directly to the end-plate receptors by iontophoresis from a m icropipette22'23 in order to test for postsynaptic effects o f lead. W e have consistently observed that lead increases the fre quency o f miniature end-plate potentials (Figs 1 and 2). The A to c M lto M M M W M W tow ito MMHimAi J Fig. 1 Effect of lead on the frequency o f miniature end-plate potentials. A , Control: the preparation was bathed in normal Ringer solution. B, Five minutes after exposing the preparation to 0.10 mM Pb2+-Ringer solution: the frequency increased greatly while the amplitudes of the miniature end-plate potentials were essentially unchanged. C, Five minutes after washing the preparation with normal Ringer solution: the frequency was no longer elevated. Oscillograms A -C were taken from the same neuromuscular junction. Vertical calibration: 1 mV. Horizontal calibration: 50 ms. NATURE VOL. 243 JUNE 8 1-S73 OOI 0.02 0 04 0 1 [Ph Fig. 2 A double logarithmic plot showing the dose-response relationship between the lead concentration and the frequency of miniature end-plate potentials. Each point is the mean s.e. for measurements made from three to five fibres. The control frequency was measured in the absence of lead and is depicted by horizontal lines (mean s.e.). All measurements were made after the preparations were exposed to the Pb2+-Ringer solution for 5 min. Although not shown in the above plot, the mean frequency decreased to 0.420.10 s_I 5 min after washing the preparations with normal Ringer solution (see also Fig. 1C). records shown in Fig. 1A were m ade while the preparation was bathed in normal Ringer solution; those in Fig. IB were made after bathing the preparation in 0.10 mM Pb2+-Ringer solution for 5 min; and those in Fig. 1C were recorded after washing with normal Ringer solution for 5 min. D ata taken from several fibres are shown graphically in Fig. 2 which is a log-log plot o f the relationship between the lead concentration and the frequency o f miniature end-plate potentials. The frequency during control conditions was 0.39 0 .2 0 s_I (mean s.e.), and it increased by almost two orders o f magnitude in the presence o f 0.10 m M Pb2+, the actual value being 22.1 7 .6 s -1. When the fibres were washed with normal Ringer solution, the frequency returned to 0 .4 2 0 .1 0 s"1 within 5-10 min. Thus, this effect o f lead is reversible (see also Fig. 1C). In 1954, del Castillo and Katz postulated that two modes o f transmitter release exist at the frog neuromuscular junction-- one for the spontaneous release o f individual quanta and another for the synchronous or phasic release o f several hundred quanta o f the transmitter, A C h 11. This hypothesis is based on the observation that magnesium did not reduce the frequency o f the spontaneous release o f transmitter even though this ion had been shown previously to inhibit the phasic release follow ing a nerve im pulse3. Calcium antagonized this blocking effect o f magnesium3, but it had no clear effect on the frequency o f miniature end-plate potentials21. Our experiments show that lead depresses the phasic release o f transmitter (Fig. 3). Sub threshold end-plate potentials were recorded in 0.8 m M C a2+/5.5 mM M g2+-Ringer solution. The m otor nerve was stimulated with pairs of pulses to facilitate transmitter release during the second transmission, thereby producing easily detectable (facilitated) end-plate potentials. The interval between pulses in a pair was 5 -7 ms, and the pair o f stimuli were applied at a rate o f 3/s. Since the amplitude o f end-plate potentials recorded from preparations bathed in high m ag nesium and low calcium Ringer solution varies according to the quantal hypothesis25, many recordings were made in a par ticular condition to obtain the m ean s.e. o f the end-plate potential. The facilitated end-plate potential during the control period was 4.71 0 .1 5 mV (n==18) (Fig. 3A). The preparation was then exposed to 0.01 m M Pb2+, the C a2+/M g 2+ ratio