Document gb75DQObvNZ8XMXDeMbbRkq9V

rwmI irr a EXHIBIT Benzene-1nduced Inhibitior??Y'P Formation in Vitro BY PETER A. DAUDU, GLENN W. GEELtiOED, MD, FACS ABSTRACT: The erythroid colony forming unit assay technique w a s used to study the inhibi-' tory effect of benzene on mouse bone marrow cell culture i n vitro. T h e ability of vitamin B, to prevent benzene-induced inhibition of ery- throid colony formation w a s also tested. At concentrations equal to o r higher than the lowest inhibitory concentration of benzene, pyridox- ine HCI significantly prevented benzene-in- duced inhibition of erythroid colony formation in vitro. However, w h e n benzene concentration was higher, the added pyridoxine HCl was inef- fective in preventing benzene-induced inhibition of erythroid colony formation in vitro, even when it w a s preincubated with the bone marr o w cells for 30 minutes prior to adding benzene. On the basis of this finding, it is proposed that benzene-induced inhibition of erythroid colony formation may involve two different mechanisms, one of which involves interaction with vitamin B6added as pyridoxine HCl. KEY INDEXING TERMS: Pyridoxine; Bone Marrow Toxicity; Anemia; Vitamin B6. [Am J Med Sci 1986;292(6):356-362.1 as in l i ~ e r ~ ' ~ d Tl sh.e~in*h~ibition in this case WN accompanied by disappearance of polyribosomes followed by the accumulation of ribosomal monomerdimers and the appearance of an intermediate elec. trophoresis peak that was previously absent. More recently, other studies have suggested thal metabolites of benzene, produced mostly by liver and bone marrow microsomal enzyme systems, are able to bind to mitochondrial DNA covalently. This covalent binding subsequently leads to inhibition d transcription and protein synthesis. The enzymelsl and/or cofactor(s) involved in the covalent bindingd these metabolites have not been identified. In the present study, we have used in vitro ery. throid colony forming unit (CFU-E)assay technique to investigate the hematopoietic toxicity of benzene and to evaluate the possibility that vitamin Bsis able to prevent benzene-induced inhibition of ery. throid colony formation by mouse bone marrow cellr in vitro. The results suggest that benzene-induced inhibition of erythroid colony formation may involve two different mechanisms. Materials and Methods Analy linckrod by maki hol. Alia eystem i tration 1. dition. 2 x 10-3, mncen t r . of the re! experimc levels of lo-', an( ments th 3 X thes no signifi lo6cultu1 3 X the mental cc Similai HC1 (Sigr prepared Ham's FNY) and Lure syste bcted fro1 duminun dutions 7 It 4C a t i Eryfhroic n plasma pith minc nl of the c The toxic effect of benzene on the hematopoietic cells has been reported by several investigators.13 These reports, which included laboratory experiments as we11 as case studies, have demonstrated that chronic benzene exposure of both animals and man leads to progressive degeneration of bone marrow, aplastic anemia, and leukemia. Although the exact mechanism by which henzene produces hematopoietic toxicity is as yet unknown, many recent studies have examined its inhibitory effects of heme synthesis.- Freedman et al' demonstrated that benzene inhibits in vitro heme synthesis at the 6-aminolevulinic acid synthetase step. Other studies have also looked at benzene inhibition of protein synthesis in reticulocytes as well From the Department of Surgery, George Washington University Medical Center, Washington,DC. Reprint requests: Dr. Glenn W . Geelhoed, 2150 Pennsylvania Avenue, NW, Washington,DC 20037. 356 Animals. The 40 mice used in this study were8 pool of control groups obtained from other experi ments conducted in the department at an earlia time; as such the mice (6-12 weeks old) consisted4 mixed strains of Swiss Webster (Balb/c NIH, ad mrimenta C57Bl.BiGW). They were maintained in a standad ndition animal care facility of the university until use. Bone Marrow Suspension. Mononuclear cells d mouse bone marrow were obtained from the two fern Aedium (c yl alcohol oral bones according to the technique described IJJ vene Oliver and G o l d ~ t e i nM. ~inimum essential medius with Hank's balanced salt solution (BSS,Grad Island Biological Company, New York, NY) supple x 10-2M x 10-JM x IO-~M x 10-5~ mented with basal medium amino acids (Microbm x 10-6M logical Associates, Bethesda, MD) was used to hw vest and wash the cells. All cell suspensions washed twice before cell counts were performed. details of the techniques used to wash, count, evaluate cell viability are as previously The final cell volume was adjusted to give 2 x 1 ~8 were prt rpf6-din this centrution c )8 concentr nucleated cells per milliliter in each case. IDecember 1986 Volume 292 Num RAMERlCAN Jt i Daudu and Geelhoed Analytical grade benzene (Thiophene free, Mal- ickrodt Chemical Works, St. Louis, MO) was used I making a 1.3M stock solution in pure ethyl alco- 11. Aliquots of this were then added to the culture - stem in microliter amounts to achieve the concenation level determined for each experimental con- tion. The concentrations tested were 1x x lov3,and 3 X M, respectively. The starting .I ncentration of 1X M was chosen on the basis the results of three separate dose-response curve periments preliminarily carried out using five vels of benzene concentration as and M, Table 1).In subsequent experi31. ents the concentration range tested was limited to r- x the starting concentration (1x M)because ?C- I significant difference in the number of CFU-Es/ l5cultured cells could be demonstrated for 2 x and at x the starting concentration under the experi- nd ental conditions described above. )le [a- of Similarly, vitamin Bs in the form of pyridoxine C1 (Sigma Chemical Company, St. Louis, MO) was epared as a 1.3 M stock solution in supplemented (8) of am's F-12 modified medium (GIBCO, New York, Y) and microliter aliquots were added to the cul- re system as needed. Vitamin B6solution was pro- w w :ne cted from direct light by wrapping the container in uminum foil. Both the benzene and the pyridoxine lutions were made up fresh every 2 weeks and kept ie ry- !lis :ed lve 4C at all times. Erythroid Colony'Cultures. The cells were cultured I plasma clots as described by Stephenson, et al" ith minor alterations. In brief, approximately 0.1 1 of the cell suspensions (approximately 2 x lo6nu- cleated cells) was pipetted into prelabeled test tubes that also contained the following ingredients: 0.3 ml Ham's F-12 modified medium (GIBCO, New York, NY), 0.1 ml, each, bovine citrated Plasma (Sigma Chemical Company), beef embryo extract (50% in Gey's BSS, GIBCO), thrombin (10 units/ml, Parke- Davis), erythropoietin (Step 111, 5.0 units/ml, Connaught Medical Res. Laboratories), and bovine serum albumin (Fraction V Powder, 10% BSA, Sigma Chemical Company). Other ingredients included in the mixture were fetal calf serum (heat inactivated, 0.2 ml), sodium bicarbonate (7.5%solution, 0.01 ml), L-glutamine (200 mM, 0.1 ml): all purchased from Microbiological Associates of Bethesda, Maryland. The plasma was added to the mixture immediately before aliquoting into the microwells. One tenth of the resulting mixture in the tube was quickly pipetted into each of three 6-mm diameter plastic microwells (Linbro Chemical Company, Inc.), allowed to clot and then incubated for 48 hours at 37C in a highly humidified environment with 5% COzand 95%air. A t the end of 48 hours, the plasma clots were re- moved from the wells (three clots per glass slide). The clots were fixed according to the technique described by Stephenson et al" with slight modifications. The clots were blotted with a 5 X 2.5-cm piece of Whatman No. 1filter paper, flattened and fixed with a 10% glutaraldehyde solution. The cells were then stained for hemoglobin using 1%solution of 3,3'-dimethoxybenzidine (Eastman Chemical Company) in methanol and counterstained with Harris' hematoxylin (Harleco, Gibbstown, NJ). To make ea xi- lier i of ind Enmental a d dition TABLE 1 CFU-E Counts/105Cultured Cells for Three Separate Dose Response Curve Experiments Expt. No. 1 Expt No. 2 Expt No. 3 Mean % of Control dedium [control] of iyl alcohol (102M] 3rna by urn and nzene I x 10-2M Ix 10-3~ Ix IO-~M ple. I x 1 0 - 5 ~ bio- I x 10-bM iar. 115 120 113 109 11 24 84 63 95 90 106 111 119 109 118 118 2 1.5 100 117 113 ? 2.3 95.8 33 23 2 6.4 19.5 71 73 -t 6.0 61.9 81 89 2 4.1 75.4 102 106 -+ 2.6 89.8 113 114 2 2.9 96.6 /era The and )SB were preliminarily camed out to establish the appropriate starting concenfration of benzene used under the assoy procedure bpted in this study. I x IO-' M was considered to be appropriate as starting concentrationin ail subsequentexperimentsbecause this ncentration appears to more closely approximate the 50%inhibiting Concentration. :d." 'he concentrationof ethyl alcohol (I x IO-' M) is the maximum concentration that was used under these experimental conditions. 10' bor 4 .AMERICANJOURNAL OF THE MEDICAL SCIENCES 357 Erythroid Colony Formation permanent slide preparations, the slides were air-dried, and cover slips applied using permount. Each clot was examined and the number of colonies containing eight or more benzidine-positive cells was counted as CFU-E's. The average number of CFU-E's from the three clots of each slide was expressed as the number of CFU-E's/105 cultured cells for the specific experimental condition per experiment. Overall, there were 32-40 experiments carried out, and in each case all the experimental conditions specified above were tested in triplicates. The student's t-test for paired and unpaired data was used to evaluate the results of all the experiments performed. AI1 calculations were carried out on a programmable hand calculator (model HP 33C). Results Figure 1A and 1B show photomicrographs (obtained at low and high magnifications, respectively) of typical erythroid colonies formed by mononuclear bone marrow cells after incubating for 48 hours a t 37C in highly humidified environment with 51 COr95% air and stained with 3,3'-dimethoxyben zidine, followed by Harris' hematoxylin countei stain. Morphologically, most of the cells have thc appearance of erythroblasts and are deeply stained A few contaminating granulocytes with typica doughnut-shaped nuclei (data not shown) could In seen in association with some of the erythroic colonies. Potentially, these contaminating celli could confound the counting process and invalidah the results. However, in the present study, the use o 3.3'-dimethoxybenzidine as stain to identify the ery throid colonies (groups of cells that appear to haw brown-orange color) greatly minimized these prob lems. Furthermore, only colonies with eight or morc benzidine-positive cells were counted as CFU-Ea. In general, the group of cells cultured in medii *that had no added benzene or pyridoxine HCl (con trol) showed a mean value ( SE)of 166 ? 5.94 CFU Es/105 nucleated cells cultured. The comparabic value for the group of cells cultured in media thal h d added pyridoxine HCl alone was 177 +: 3.21 T .is suggests that added pyridoxine alone at a con ientration of 1x M improved erythroid colon] formation by ever, is not st. Conversely dia that had a I x 10-3 M sh Es/106 cells 8 lower (by 37.: trol (t = 20.14 : Similarly, cells culturec is significant CFU-Es/105c bone marrow benzene and tration of p c 0.001). TI have providc induced inhi under these e Figure 2 sk of erythroid ( cultured in T 3 x 10-3 M t constant con , i ? 0L , '* I + ,. , *(I 3 ," + c 4 " ";s b Figure 1A. Photomicrograph of erythroid colonies after 48 hours incubation. Erythroid colony cells were stained wlm 3.3'-dimethoxybenzidineand counter stained with Harris' hematoxylin. Under this condition cells with hemoglobin show brown-orange color and only colonieswith eight or more such cells were counted as CFU-Es (originalmagnification. x 200) 358 December 1986 Volume 292 Numbub igure 1B. A rep! ixperlmentalco HE AMERICAN JOU . Daudu a n d Geelhoed 1% ormation by 6.6 2 0.12%. This improvement, how- n- :ver, is not statistically significant. er Conversely, the group of cells cultured in the me- .he lia that had added benzene alone at concentration of .d Ix M showed a mean value of 62 ? 1.99 CFU- .a1 be lid M105 cells cultured. This value is significantly ower (by 37.3 2 2.01) than the mean value for con- .rol(t= 20.149, p < 0.001). Ile Similarly, the mean value (62 ? 1.99 CFU-Es) for ta cells cultured in the presence of 1x M benzene of is significantly lower than the mean value (99 2 5.40 'P ve CFU-Es/105cultured cells) obtained for bone marrow cells cultured in medium the group of with added Ib- benzene and pyridoxine together at equal concen- re tration of 1X M, respectively (t= 8.162, p < 0.001). This suggests that pyridoxine HCl may !ia have provided some protection against benzene- n- induced inhibition of erythroid colony formation U- under these experimental conditions. ,le Figure 2 shows graphically the percent inhibition at :1. n- of erythroid colony formation by bone marrow cells cultured in media that had 1x 2 x loe3 and 3 x M benzene added simultaneously with a constant concentration (1x M) of pyridoxine 2Y HC1 in each case. Overall, the inhibition of erythroid colony formation increased (from 36.4 2 1.99% to 53.8? 2.5% and 54.4 2 3.84%, respectively) as the concentration of benzene increased from 1x M to 2 x M and 3 x M, respectively in spite of the added (1x M)pyridoxine in each case. The difference between the percent inhibition at 2 x l o w 3 M and 3 x M benzene is not statistically signifi- cant. However, the difference between percentage inhibition at 1x M and a t 3 x M benzene was statistically significant (t= 3.992, p < 0.001). The group of bone marrow cells cultured in media that had pyridoxine added at a concentration of 2 x M ,and 3 x M in the presence of a constant amount (1x M) of benzene showed 16.52 0.53% and 24.82 0.71% inhibition respec- tively. Again, the differences between the percent inhibition a t the two experimental conditions are not statistically significant. To test for possible benefit of preincubation of bone marrow cells with pyridoxine, the cells were first incubated for 30 minutes in culture media that con- tained 1x M pyridoxine HCl and then cultured for 48 hours. x/ d 4 'Ir W Ih W )I. lgure 16. A representative erythroid colony at higher rnagniflcatlon to show typical appearance of the cells under our txperlmentalcondition (originalmagnification x 1000). 16 HE AMERICAN JOURNAL OF THE MEDICAL SCIENCES 359 Eryfhroid Colony Formation 80 70 60 50 -e-J..cC0--z 40 a-" 30 _ _ increasing Pyridoxine HCI - increasing Benzene T \ \ '\ 20 10 0 I 11 1 .o 2.o 3.0 Additive (x M) Figure 2. Percentage inhibition of erythroid colony formation under increasing pyridoxine (solid line) and increasing benzene concentration (broken lines). To correct for the effect of p. y. ridoxine alone, % Inhibition was calculated as ) 3x pyridoxine xioo. Figure 3 shows a histograph of the mean values ( -+ SE) of CFU-Es/105cells cultured (aspercent control) under these experimental conditions. Overall there is no statistically significant difference .between the mean values of CFU-Es/105 cells determined for the group of bone marrow cells cultured in media which had benzene added simultaneously with pyridoxine and the mean values obtained for the cells preincubated with pyridoxine at all three levels of benzene concentration tested. Discussion The erythroid colony forming unit assay technique is based on the principle that the number of colonies formed is directly proportional to the number of nu- cleated marrow cells incubated in the individual plasma clot. The data presented in this study are for tests carried out on the bone marrow cells obtained from 40 mice. The data indicate that added benzene a t a concentration of 1x M significantly inhibits erythroid colony formation under the in vitro conditions described. When pyridoxine HC1 is added together with benzene at equal concentration (1x M each) or higher than benzene (2 x M pyridoxine, respec. tively, added with 1x M benzene), there was significant protection against benzene-induced inhibition of erythroid colony formation as indicated by the significant increase in the number of CFU-Ed lo5 cells cultured under this condition. However, when the concentration of pyridoxine is maintained at 1x M while the concentration of benzene is increased to 2 x and 3 x M, respectively, the added pyridoxine was ineffective in preventing benzene-induced inhibition of erythroid colony formation. Furthermore, preincubation of the bone marrow cells with pyridoxine HCl (at 1X M)for Pyridoxine HCI & Benzene added 0Simultaneously Pre-incubated with Pyridoxine HCI then added Benzene 110- 100- 90- 80- 70- 60- -50 -4 0 30- -20 m10AsL 0 1.o 2.0 3.0 9Fi ure 3. A histographof the mean values ( 5 SE) of CFU-Epl 10 cells cultured under added pyridoxine and benzenob muitaneousiy, and added pyridoxine 30 minutes prim b' adding benzene. Preincubation with pyridoxine did nb ,.provide added protection against benzene inhibllim$ erythroid colony formation. 30 minutes 3 x 10-3 M, protection : erythroid co In genera inhibits eryf '.with similai gators.'. l3 Since the studied the ing the sub studies havc anism by v toxicity. Mo: marrow the (pronormobl line)are the toxicity.'. At the mi demonstrak lites may be is suggested diate benze direct intera somes of the arrest of ma hibition of km.16, 17 However, benzene (pos lites) suppre but also inhi thesis in dip and rabbit 1 have reportt amino levul benzene-indL reticulocytes late that th induced inhi, the ALA syi etrated that ; ulocyte susp rsnd reversed ration into erne synthe presented th: uct(s) of ben: covalently; s benzene-indu In the pre: Bynthesis wa. metabolites c DNA and/or I observed deci the experime of the data o mperimen ts, 360 December1986 Volume 292 NU-) #AMERICAN JOU Daudu and Geelhoed ial 10 minutes prior to adding benzene (at 2 x and or I x M, respectively) did not result in improved ed )rotection against benzene-induced inhibition of ne lrythroid colony formation. in- In general, the finding that benzene significantly .ro nhibits erythroid colony formation is in occordance vith similar data reported by several other investi:n- ;ators.7.l'L.13 or Since the classical experiment of Selling,14which X- jtudied the sequence of hemopoietic changes follow'aa ing the subcutaneous injection of benzene, several hi- studies have been conducted to evaluate the mechby anism by which benzene produces hematopoietic <s/ toxicity. Most of these studies agree that in the bone er, marrow the cells in early stages of development :ed pronormoblasts and normoblasts of the myeloid ia line)are the most sensitive to benzene hematopoietic lY, toxicity.', l5 ng At the molecular level, experimental data have or- demonstrated that one or more of benzene metabone lites may be involved in its hematopoietic toxicity. It for is suggested that the putative metabolite(s) may me- diate benzene-induced hematopoietic toxicity by direct interaction with the nucleus and the chromosomes of these cells. This in turn could result in the arrest of maturation of bone marrow cells and/or in- I hibition of cell division in the erythrocytic sys- However, other studies have demonstrated that benzene (possibly through the action of its metabo- lites) suppresses not only DNA and RNA synthesis, but also inhibits heme and cytoplasmic protein syn- lhesis in differentiating bone marrow cells of mice and rabbit reticulocytes respectively. Forte et a14 have reported data that demonstrate that delta- I amino levulinic acid (ALA) or hemin reversed benzene-induced inhibition of protein synthesis in reticulocytes. This has led F'reedman et al' to postulate that the specific molecular site of benzeneinduced inhibition of heme synthesis is at or before the ALA synthetase step. The group also demonstrated that 1mM pyridoxine added to a rabbit reticulocyte suspension significantly protected against and reversed benzene inhibition of 14C-glycineincorporation into haemin that was used as a n index of heme synthesis. More recently, evidence has been presented that suggests that some metabolic product(s) of benzene may bind to mitochondrial DNA covalently; such covalent binding could lead to benzene-induced inhibition of protein synthesis." w In the present study, it is possible that protein synthesis was inhibited by covalent binding of the metabolites of the added benzene to mitochondrial DNA and/or RNA; this in turn could have led to the e& loj r nOl n of observed decrease in the number of CFU-Es under the experimental condition described. On the basis of the data observed for the added pyridoxine HC1 experiments, however, it appears that there may be an additional mechanism by which benzene inhibits erythroid colony formation in vitro. It appears also that this other mechanism operates at a moderate (1x M) level of benzene concentration and could be prevented by the added pyridoxine HCl. Several studies have been reported that demon- strate the regulatory role of vitamin B6 (as pyri- doxal-5-phosphate) in heme and protein synthes ~ s . ' ~T.h~e"protective effect of the added pyridoxine in the present study may be due to its regulatory role in heme and protein syntheses. Under these conditions, added pyridoxine HC1 should prevent benzeneinduced inhibition of erythroid colony formation at all levels of benzene concentration. However, the preceding data appear to suggest otherwise. The data reported in this study have not fully explained all of the known effects of benzene on hemopoietic systems. Nevertheless, they do support the view that one of the effects of benzene apparently involves the inhibition of erythroid colony formation in vitro. In addition, the data suggest that the apparent benzene-induced inhibition of erythroid colony formation may involve two different mechanisms. However, it would be interesting if these findings are investigated further in more primitive precursor cells such as the erythyroid burst forming cells (BFU-Es)and in vivo experiments to investigate further the relationship between the two mechanisms and the possible involvement of vitamin B6. References 1. Kalf GF, Rushmore T, Synder R Benzene inhibits RNA syn- thesis in mitochondria from liver and bone marrow. Chem Biol Interact 42:353,1982. 2. Synder R, Lee EW, Kocsis JJ, Witmer CM: Bone marrow depressant and leukemogenic actions of benzene: Mini review. Life Sci 21:1709-1722, 1977. 3. Rosenthal CJ,Synder CA: The effects of ethanol and the role of the spleen during benzene-induced hematotoxicity. Toxicology 30:283,1984. 4. Fortes FJ, Cohen HS, Rosman J,Freedman ML: Hemin rever- sal of benzene-induced inhibition of reticulocytes protein synthesis. Blood 47:145-154. 1976. 5. Wildman JM, F'reedmanML, Rosman J, Goldstein B: Benzene and lead inhibition of rabbit reticulocyte heme and protein synthesis: Evidence for additive toxicity of these two components of commercial gasoline. Res Commun Chem Pnthol Pharmacol 13:473-488,1976. 6. Lee EW, Kocsis J, Synder R Dose dependent inhibition of "Fe incorporation into erythrocytes after a single dose of benzene. Res Commun Chem Pathol Pharmacol 5:547-550,1973. 7. F'reedman ML, Wildman JM, Rosman J, Eisen J,Greenblatt DR: Benzene inhibition of in vitro rabbit reticulocyte haem synthesis at delta aminolaevalinic and synthetase: Reversal of benzene toxicity by pyridoxine. Br J Haemntol 35:49-60, 1977. 8. Tyfriates GP, Choulis NH: Effect of benzene on rat liver polyribosomes. Biochem Pharmacol20:1669-1677,1972. 9. Oliver JP, Goldstein AL: Rapid method of preparing bone marrow cells from small laboratow animals. J Immunol Meih 19:289-292. 1978. 10. Daudu PA. Geelhoed GW: Numbers of T-cell rosettes in the peripheral' blood of patients receiving maintenance hemodialysis. South Med J 75570, 1982. b U 4 XAMERICAN JOURNAL OF THE MEDICAL SCIENCES 361 I .rsnr.... ..^^..^^, EiythroidColony Formation 11. Stephenson JR, Axelrad AA, McLeod DL. Shreeve MM: Induction of colonies of hemoglobin-synthesizing cells by erythropoietin in vitro. Proc Nafl Acad Sci USA 6815421546, 1971. 12. Gill DP, Jenkins VK, Kempen RR, Ellis S The importance of DluriDotentiaI stem cells in benzene toxicity. Toxicology i6:1&-171,1980. 13. Moriyama Y, Fisher JW: Effect of kstostero,ne and erythro- poient in on erythroid colony formation in human bone mar- row cultures. Blood 45:665-670,1975. 14. Selling L: Benzol as a leucotoxin: Studies on the degeneration and regeneration of blood and hemopoietic organs. John Hopkins Hosp Rep 17:83-142.1916. 15. Moeschlin S, Speck B: Experimental studies on the mechanism of action of benzene on the bone marrow (radioautograph studies using 'H-thymidine). Acfa Haematol 38:lOP 111.1967. 16. Rusch GM, Leong BKJ, Laskin S: In vitro benzene metab lism, in Laskin S,Goldstein B (eds):Benzene Toxicity a Crib ical Eualuation. New York: McGraw-Hill, 1977. 17. Bolcsak LE, Nerland EE: Inhibition of erythropoiesis by bene zene and benzene metabolites. Toxicol Appl Pharmad 69:363,1983. 18. Rushmore T, Synder R, Kalf T Covalent binding of benzene and its metabolites to DNA in rab'oit bone marrow milo. chondria in vitro. Chem Biol Intemct 49133,1984. 19. Schulman MP, Richert DA: Heme synthesis in vitamin b a n d pantothenic acid deficiencies. J Biol Chem 226:181,1957. 20. GraeaoJ, Hines J D A comparative electron microscopic study of refractory and alcoholic sideroblastic anemias. Br J Hor. mafoll735,1969. Sponr Side BY ROBEF CHARLES ABSTRAC' tion within Lo intrave bubbles. 11 istration o mechanism Ipontaneot have been tource of smotely in !irculation mce. Two I mtly spont; teart cham ieart failur nonary hyl nechanism lomenon rc ailure and 'ERMS: Sp tight Heal Taphy. [An &;go; * eart chambc eports have etail. The I escribe two pontaneous lght heart irdiography, )sewation, i ation for the chnlques an The echoes ere intenselj ineously in 362 From the Depar, wpital of Baltim Reprint request: w, Sinai Hospitc December1986 Volume 292 Number4 AMERICAN JOUR