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Percutaneous Absorption of Mercury in Man
Vol. 50, No. 4
THE JOURNAL OF INVESTIOATIVE DERMATOLOGY
Copyright
Printed in U.S.A.
1968 by The Williams & Wilkins Co.
PERCUTANEOUS ABSORPTION OF MERCURY IN MAN 1. A STUDY BY ELECTRON MICROSCOPY OF THE PASSAGE OF TOPICALLY APPLIED MERCURY SALTS THROUGH STRATUM CORNEUM OF SUBJECTS NOT SENSITIVE TO MERCURY*
INGA SILBERBERG, M.D.
This paper presents part of a continuing hyde (4%) with aqueous sodium cacodylate study of the transit of mercury through the (0.1M) as a buffer (pH 7.4). They were then
in two changes of cold isotonic saline for human skin. This report concerns the passage washed 45 minutes and two variations of further processof mercury through the stratum eorneum. ing were made, namely, (a) some pieces of each Mercuric sulfide is sufficiently dense to kind were immersed in an aqueous solution of scatter electrons and can easily he dis- ammonium sulfide (1%) at 4° C for 10 minutes; tinguished from other substances at the and (b) other pieces, as controls, were placed in
distilled water at 4° C for 10 minutes. Subultrastruetural level. Thus, it is a substance sequently all specimens were washed for 3 minutes Which can be, and has been, used as a tracer in three changes of cold isotonic sahne. Finally in studies by electron microscopy (1, 2). Also,
they were all dehydrated in graded alcohols and
it has been shown that mercuric chloride embedded in Maraglas after the method of Free-
and Spurloek (4). and ammoniated mercury in the presence of man 2. Another set of specimens was first treated ammonium sulfide are converted into mercuric in the above manner except that before desulfide (3). All of these facts were utilized in hydrating and embedding in Maraglas, the pieces developing a method for visualizing mercury were further fixed for one hour in an aqueous solu-
tion of osmium tetroxide (1%) with aqueous so-
in skin.
dium phosphate (0.lM) as a buffer (pH. 7.4). 3. A third set of specimens was fixed in a mix-
ture of glutaraldehyde and osmium tetroxide according to the method of Trump and Bulger (5).
METHOOS AND MATERIALS
Experiments were performed on two male vol-
During dehydration some of the specimens were
unteers. In one subject an aqueous solution of placed in a 1% solution of phosphotungstic acid in mercuric chloride (0.1%) was applied to a site on
absolute ethanol for one hour.
Sections silver to gold in color were cut with a the back and distilled water was applied to a paired site. In the other subject ammoniated microtome from all specimens processed in all
mercury in white petrolatum (5%) was applied to the above manners and placed on 200 mesh grids one site on the back and white petrolatum alone without supporting film. Before examination of in approximately equal amount to a paired site. them, some were stained with a solution of uranyl The skin sites that were prepared appeared to acetate (1% in absolute alcohol) and/or Reynold's
be normal in both subjects, and simultaneous
patch tests with mercury salts to other sites were negative.
lead citrate (6); others were left unstained. The stained specimens were treated with the same batch of stain. Carbon was layered on all of them by evaporation on both sides of the grid. Four
Two hours after application of the test materials, punch biopsies were taken from all sites areas from four slices from each specimen, finished under local anesthesia with lidoeaine® hydro- in the manners described, were examined. In addition, tissue taken from one of the volunchloride. The specimens were diced into cubes measuring about 1 mm3. Fixation of the bits of teers, from a site not treated with mercury, was processed for electron microscopy as described tissue was then done in three ways as follows: 1. Several specimens from both the mercury- above, up to the point when sections were on treated and control sites were immersed for two the grids and ready for staining. They were then hours at 4° C in an aqueous solution of glutaralde- immersed in a 0.05% aqueous solution of mercuric chloride for two minutes and washed for three minutes in three changes of distilled water. Fol* From the Department of Dermatology of lowing that, some were coated with carbon; others New York University School of Medicine, New were further immersed in a 1% aqueous solution York City, New York. ammonium sulfide for two minutes, again This work was supported in part by USPHS, of washed in three changes of distilled water for General Research Support Grant FR-05399. three minutes and finally coated with carbon. All tissues were examined and photographed in a Siemens Elmiskop I.
Accepted for publication October 27, 1967.
The author wishes to acknowledge with thanks
the technical help of Mr. Thomas M. Valentine.
323
324
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY RESULTS
The findings in this study are pictured in the accompanying figures. Figures 1, 2 and 3 are of representative cell layers of the stratum eorneum from its uppermost surface down-
In Fig. 3 the middle to the lowest layers of the stratum eorneum from the same tissue as that in Figs. 1 and 2 are depicted. Electrondense particles (p) are present in or overly-
ing the cell layers of the stratum corneum. These particles are especially prominent in wards towards the stratum granulosum. the intercellular spaces. In some areas they Figure 1 shows a section from skin to are more numerous in the region of the deswhich mercuric chloride had been applied. mosomes. In some cell layers electron-dense From above downward, one sees aggregated bodies (x) of unknown nature are present. electron-dense particles (F) apparently upon Fig. 4 shows an unstained section of tissue the surface of the stratum eorneum. Overlying taken from a site of skin to which mercuric or within a superficial cell of the stratum chloride had been applied. The intercellular eorneum dispersed electron-dense particles space (IC) contains electron-dense bodies (p) are seen. These are especially prominent in the area of desmosomes. Both types of with areas of greater electron density in or on them (1). These aggregates were occasionally particles (F, p) are taken to represent mercury seen in stained sections of skin to which the in various complexings. The intercellular vehicle alone bad been applied but in that space (IC) contains some other electron- case they had quantitatively fewer electrondense particles (Y) in aggregations. These dense particles associated with them than are
latter aggregates may occasionally he seen in found here. Fartieles (p) are especially promstained sections of skin to which no mercury salt had been applied. In the intercellular inent in regions of desmosomes. Areas of in-
electron-density are found in some space between the first (C 1) and second creased portions of the membrane (M) of cells of the (C 2) layers of the stratum eorneum there is evidence of a moderately electron-dense, intercellular substance with areas of variable and lesser opacity than the surrounding milieu, as well as some vesicular structures. This was seen with equal frequency in control skin to
stratum eorneum. Some of these electron-dense
areas (M) have particles (p) in or on them. These particles represent mercury in various
complexes. In skin sites treated with the
vehicle alone one surface of a membrane of a cell of the stratum corneum occasionally shows which the vehicle alone had been applied. Figure 2 shows an area from the same speci- increased electron-density. In no instance were men subjacent to that seen in Fig. 1. Electron- there particles (p) present in these loca-
dense aggregates marked Y are especially tions. Figure 5 is the same as Fig. 4, except it prominent at the top and bottom of the secshows with more clarity the position of parond intercellular space (IC 2). It is difficult to say if mercury or its complexes are within ticles (p) at desmosomal junctions. Figure 6 shows an unstained section of noror upon these aggregates because appearances of this sort are occasionally seen in stained mal human skin which was immersed in mersections of stratum eorneum of skin treated curic chloride, washed in distilled water and with the vehicle alone.
coated with carbon. Variation in sizes of
CODE OF LETTERS USED IN LABELING TO FHOTOMICROCRAFHS
C Cell layer of stratum corneum D, d Desmosome IC Intercellular space
Electron-dense portion of a membrane of a cell of the stratum corneum Areas of variable and lesser opacity in the intercellular substance F, p Electron-dense particles which are taken to represent mercury in various complexings v Vesicular structures in the intercellular space X, x Electron-dense bodies of unknown nature found in some of the cell layers of the stratum corneum Y Aggregates of electron-dense particles which are also occasionally seen in stained sections of skin to which no mercury salt had been applied. There is, however, a recognizable increase of electron-density in those sites which were treated with the mercury M
0
salt.
325
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ABSORPTION OF MRRCIJRY
FIG. 1. Stained section of skin to which mercuric chloride had been applied. See text for explanation. X43,000. The insert demonstrates the various sizes of the particles seen. Stained with uraoyl acetate and lead citrate. X215,000.
mercury particles (P, p) is seen. The prti- (IC). Unstained sections of normal skin that cles are randomly distributed. An electron- were immersed in mercuric chloride, treated dense aggregation (Y) as well as areas of lesser
opacity (0) are present in intercellular spaces
with ammonium sulfide, washed in distilled water and coated with carbon were also ex-
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
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FIG. 2. Section of skin to which mercuric chloride had been applied. Stained with
uranyl acetate and lead citrate. X47,500.
amined. The number of particles was roughly
equal and showed similar variations in size as those shown in Figure 6.
moniatcd mercury ointment, cnn be visualized by electron microscopy in its passage through the cellular layers and intercellular spaces cf the stratum corneum. The sizes of
It was found that with proper processing, mercury, when applied topically either as a electron-dense particles as mercury salts or as
solution of mercuric chlcride or as am- complexes of mercury with protein visible on
ABSORPTION OF MERCURY
327
Fm. 3. This photomicrograph depicts skin treated with mercuric chloride. The area is from middle to lowest layers of the stratum corneum from the same tissue as that depicted
in Figures 1 & 2. In some cell layers electron-dense bodies (x) of unknown nature are present. Stained with uranyl acetate and lead citrate. X43,300.
electron photomierographs were found to vary in glutaraldebyde, treated with ammonium
sulfide and postfixed in osmium tetroxide (Figures 1—5). The use or omission of 1% complexes was obtained when tissue was fixed phosphotungstic acid did not influence the from 50 A to 350 A (see Fig. 1).
The best visualization of mercury or its
328
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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
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e
4,
Ce
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Fm. 4. Unstained section of tissue taken from a site of skin to which mercuric chloride had been applied. X30,000.
visualization of the electron-dense particles. to be fewer in number when compared to When specimens of skin from patients that specimens that were dipped in ammonium were treated with a mercurial were not sulfide. This was also true of the tissue fixed dipped in ammonium sulfide some electron- in a mixture of glutaraldebyde and osmium dense particles were present, but they seemed tetroxide or in glutaraldebyde alone. The
ABSORPTION OF MERCURY
FIG.
329
5. Unstained section of stratum corneum from a site where mercuric chloride had
been applied. X50,000.
procedure of carbon coating both sides of the curio sulfide (2). They (P, p) represent either grid tended to minimize migration or subli- mercuric sulfide with or without a surroundmation of mercury in the tissues (7). Only those areas of the skin which had been
ing coat of protein, or mercury particles
which have complexed with tissue proteins, or treated with mercury salts and not the control are particles in aggregation, or combinations
sites treated with the vehicles alone showed of these possibilities. Evidence that they are the presence of the electron-dense particles de- specific precipitates is the following: 1. They scribed above. The unstained sections showed are found only in those sites where mercury the presence of electron-dense particles with salts were applied topically. 2. The electron-
about the same distribution as that of the dense particles are not seen in specimens stained tissue (Figures 4, 5). Occasionally from areas of skin treated with the vehicle osmium tctroxidc fixation resulted in some alone, but processed in an identical manner. black precipitates but, after washing with saline 3. The particles arc seen in unstained as well only minimal amounts were present. as stained tissue. Staining was used to in-
crease contrast, thereby facilitating precise localization of the particles as well as faciliParticles designated P, p on the accom- tating focusing at higher magnifications. 4. panying photomicrographs appear to be some The particles localize in certain definable form of mercury. The range of sizes of the areas and are not diffusely scattered as though tarticles described is similar to that of mer- smeared over the tissue section. 5. They are DIscussION
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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
330
Ftc. 6. Unstained section of normal human skin which was immersed in mercuric
chloride, washed in distilled water and coated with carbon. Variation in size of mercury particles (P, p) is seen. The particles are randomly distributed. X60,000.
found as small aggregates at the outermost Recently Lagerholm and Frithz (8) desurfaces of the stratum corneum, within or scribed the intercellular distribution of meroverlying some but not all cell layers of the cury compound in the epidermis of patients stratum corneum, at desmosomal junctions and with psoriasis. An increase in electron density in the intercellular substance,
of ribosomes, of ehromatin material and of
331
ABSORPTION OF MERCURY
certain intranuclear particles was found in complexes which have migrated through the cells of psoriatic epidermis to which am- stratum corneum by way of the intercellular moniated mercury ointment had been applied
spaces in a maze-like pattern as well as
in vivo.
through the cell layers.
reach their locations in the tissue sections.
Further work of this nature is in progress in the hope that this system can be used to
Several namely:
themselves,
study percutaneous absorption of mercury in human subjects with allergic eczematous con-
1. During the process of cutting sections the cutting instrument may drag mercury
tact dermatitis due to mercury compounds
within five minutes after application of
REFERENCES
The question arises as to how the particles possibilities
suggest
and absorption of other metals which can be along the surface of the specimen. To test this visualized in a comparable manner. possibility specimens which had been taken
mercury salts were cut and examined. These 1. Alkane, J. F.: The passage of colloidal particles specimens did not show electron-dense paracross the dermal capillary wall under the influence of histamine. Quart. J. Exp. Physticles such as those interpreted here as being mercury or its complexes.
2. During the process of fixation of the specimens the particles are moved so that they appear to be lying in or on the stratum
iol., 44: 51, 1959.
2. Majno, G. and Palade, G. E.: Studies on inflam-
mation. I. The effect of histamine and serotonin on vascular permeability: An electron microscopic study. J. Biophys. & Biochem. Cytol., 11: 571, 1961.
corneum cells. This possibility cannot be ex- 3. Whitmore, F. C.: Organic Compounds of Mercury. Chemical Catalog Co., 50, 1921. cluded. However, four tissue slices from each 4. Freeman, J. A. and Spurlock, B. 0.: A new epoxy embedment for electron microscopy. specimen were processed simultaneously and J. Cell. Biol., 13: 437, 1962. sections from each slice were cut. The loca- 5. Trump, B. F. and Bulger, R. E.: New ultration of the particles in the sections from structural characteristics of cells fixed in a
various tissue slices was similar. This sugglutaraldehyde-osmium tetroxide mixture. Lab. Investig., 15: 368, 1966. gests that the sites of localization are not ac- 6. Reynolds, E. S.: The use of lead citrate at high cidental.
3. There is actual transcellular or intercellu-
pH as an electron-opaque stain in electron microscopy. J. Cell. Biol., 17:
208, 1963.
F. A. and Finck, H.: The use of specific lar migration in some layers of the stratum 7. Pepe, antibody in electron microscopy. II. The vis-
corneum. Figures 1, 2, 3, and 5 are most suggestive of this possibility.
All of the results obtained suggest that there
is specific localization of particles (F, p) in sections of skin to which mercury salts had been applied. The particles represent mercury
ualization of mercury-labeled antibody in the electron microscope. J. Biophys. & Biochem. Cytology, 11:
521, 1961.
8. Lagerhoim, B. and Frithz, A.: Cellular changes in the psoriatic epidermis. V. The submicro-
scopic intracellular distribution of mercury compound in the psoriatic epidermis. Act. Derm. Ven., 47: 222, 1967.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.
THE JOURNAL OF INVESTIOATIVE DERMATOLOGY
Copyright
Printed in U.S.A.
1968 by The Williams & Wilkins Co.
PERCUTANEOUS ABSORPTION OF MERCURY IN MAN 1. A STUDY BY ELECTRON MICROSCOPY OF THE PASSAGE OF TOPICALLY APPLIED MERCURY SALTS THROUGH STRATUM CORNEUM OF SUBJECTS NOT SENSITIVE TO MERCURY*
INGA SILBERBERG, M.D.
This paper presents part of a continuing hyde (4%) with aqueous sodium cacodylate study of the transit of mercury through the (0.1M) as a buffer (pH 7.4). They were then
in two changes of cold isotonic saline for human skin. This report concerns the passage washed 45 minutes and two variations of further processof mercury through the stratum eorneum. ing were made, namely, (a) some pieces of each Mercuric sulfide is sufficiently dense to kind were immersed in an aqueous solution of scatter electrons and can easily he dis- ammonium sulfide (1%) at 4° C for 10 minutes; tinguished from other substances at the and (b) other pieces, as controls, were placed in
distilled water at 4° C for 10 minutes. Subultrastruetural level. Thus, it is a substance sequently all specimens were washed for 3 minutes Which can be, and has been, used as a tracer in three changes of cold isotonic sahne. Finally in studies by electron microscopy (1, 2). Also,
they were all dehydrated in graded alcohols and
it has been shown that mercuric chloride embedded in Maraglas after the method of Free-
and Spurloek (4). and ammoniated mercury in the presence of man 2. Another set of specimens was first treated ammonium sulfide are converted into mercuric in the above manner except that before desulfide (3). All of these facts were utilized in hydrating and embedding in Maraglas, the pieces developing a method for visualizing mercury were further fixed for one hour in an aqueous solu-
tion of osmium tetroxide (1%) with aqueous so-
in skin.
dium phosphate (0.lM) as a buffer (pH. 7.4). 3. A third set of specimens was fixed in a mix-
ture of glutaraldehyde and osmium tetroxide according to the method of Trump and Bulger (5).
METHOOS AND MATERIALS
Experiments were performed on two male vol-
During dehydration some of the specimens were
unteers. In one subject an aqueous solution of placed in a 1% solution of phosphotungstic acid in mercuric chloride (0.1%) was applied to a site on
absolute ethanol for one hour.
Sections silver to gold in color were cut with a the back and distilled water was applied to a paired site. In the other subject ammoniated microtome from all specimens processed in all
mercury in white petrolatum (5%) was applied to the above manners and placed on 200 mesh grids one site on the back and white petrolatum alone without supporting film. Before examination of in approximately equal amount to a paired site. them, some were stained with a solution of uranyl The skin sites that were prepared appeared to acetate (1% in absolute alcohol) and/or Reynold's
be normal in both subjects, and simultaneous
patch tests with mercury salts to other sites were negative.
lead citrate (6); others were left unstained. The stained specimens were treated with the same batch of stain. Carbon was layered on all of them by evaporation on both sides of the grid. Four
Two hours after application of the test materials, punch biopsies were taken from all sites areas from four slices from each specimen, finished under local anesthesia with lidoeaine® hydro- in the manners described, were examined. In addition, tissue taken from one of the volunchloride. The specimens were diced into cubes measuring about 1 mm3. Fixation of the bits of teers, from a site not treated with mercury, was processed for electron microscopy as described tissue was then done in three ways as follows: 1. Several specimens from both the mercury- above, up to the point when sections were on treated and control sites were immersed for two the grids and ready for staining. They were then hours at 4° C in an aqueous solution of glutaralde- immersed in a 0.05% aqueous solution of mercuric chloride for two minutes and washed for three minutes in three changes of distilled water. Fol* From the Department of Dermatology of lowing that, some were coated with carbon; others New York University School of Medicine, New were further immersed in a 1% aqueous solution York City, New York. ammonium sulfide for two minutes, again This work was supported in part by USPHS, of washed in three changes of distilled water for General Research Support Grant FR-05399. three minutes and finally coated with carbon. All tissues were examined and photographed in a Siemens Elmiskop I.
Accepted for publication October 27, 1967.
The author wishes to acknowledge with thanks
the technical help of Mr. Thomas M. Valentine.
323
324
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY RESULTS
The findings in this study are pictured in the accompanying figures. Figures 1, 2 and 3 are of representative cell layers of the stratum eorneum from its uppermost surface down-
In Fig. 3 the middle to the lowest layers of the stratum eorneum from the same tissue as that in Figs. 1 and 2 are depicted. Electrondense particles (p) are present in or overly-
ing the cell layers of the stratum corneum. These particles are especially prominent in wards towards the stratum granulosum. the intercellular spaces. In some areas they Figure 1 shows a section from skin to are more numerous in the region of the deswhich mercuric chloride had been applied. mosomes. In some cell layers electron-dense From above downward, one sees aggregated bodies (x) of unknown nature are present. electron-dense particles (F) apparently upon Fig. 4 shows an unstained section of tissue the surface of the stratum eorneum. Overlying taken from a site of skin to which mercuric or within a superficial cell of the stratum chloride had been applied. The intercellular eorneum dispersed electron-dense particles space (IC) contains electron-dense bodies (p) are seen. These are especially prominent in the area of desmosomes. Both types of with areas of greater electron density in or on them (1). These aggregates were occasionally particles (F, p) are taken to represent mercury seen in stained sections of skin to which the in various complexings. The intercellular vehicle alone bad been applied but in that space (IC) contains some other electron- case they had quantitatively fewer electrondense particles (Y) in aggregations. These dense particles associated with them than are
latter aggregates may occasionally he seen in found here. Fartieles (p) are especially promstained sections of skin to which no mercury salt had been applied. In the intercellular inent in regions of desmosomes. Areas of in-
electron-density are found in some space between the first (C 1) and second creased portions of the membrane (M) of cells of the (C 2) layers of the stratum eorneum there is evidence of a moderately electron-dense, intercellular substance with areas of variable and lesser opacity than the surrounding milieu, as well as some vesicular structures. This was seen with equal frequency in control skin to
stratum eorneum. Some of these electron-dense
areas (M) have particles (p) in or on them. These particles represent mercury in various
complexes. In skin sites treated with the
vehicle alone one surface of a membrane of a cell of the stratum corneum occasionally shows which the vehicle alone had been applied. Figure 2 shows an area from the same speci- increased electron-density. In no instance were men subjacent to that seen in Fig. 1. Electron- there particles (p) present in these loca-
dense aggregates marked Y are especially tions. Figure 5 is the same as Fig. 4, except it prominent at the top and bottom of the secshows with more clarity the position of parond intercellular space (IC 2). It is difficult to say if mercury or its complexes are within ticles (p) at desmosomal junctions. Figure 6 shows an unstained section of noror upon these aggregates because appearances of this sort are occasionally seen in stained mal human skin which was immersed in mersections of stratum eorneum of skin treated curic chloride, washed in distilled water and with the vehicle alone.
coated with carbon. Variation in sizes of
CODE OF LETTERS USED IN LABELING TO FHOTOMICROCRAFHS
C Cell layer of stratum corneum D, d Desmosome IC Intercellular space
Electron-dense portion of a membrane of a cell of the stratum corneum Areas of variable and lesser opacity in the intercellular substance F, p Electron-dense particles which are taken to represent mercury in various complexings v Vesicular structures in the intercellular space X, x Electron-dense bodies of unknown nature found in some of the cell layers of the stratum corneum Y Aggregates of electron-dense particles which are also occasionally seen in stained sections of skin to which no mercury salt had been applied. There is, however, a recognizable increase of electron-density in those sites which were treated with the mercury M
0
salt.
325
k..
pop
ABSORPTION OF MRRCIJRY
FIG. 1. Stained section of skin to which mercuric chloride had been applied. See text for explanation. X43,000. The insert demonstrates the various sizes of the particles seen. Stained with uraoyl acetate and lead citrate. X215,000.
mercury particles (P, p) is seen. The prti- (IC). Unstained sections of normal skin that cles are randomly distributed. An electron- were immersed in mercuric chloride, treated dense aggregation (Y) as well as areas of lesser
opacity (0) are present in intercellular spaces
with ammonium sulfide, washed in distilled water and coated with carbon were also ex-
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
t
4
PS
a)
"ta4t!
Ti
326
FIG. 2. Section of skin to which mercuric chloride had been applied. Stained with
uranyl acetate and lead citrate. X47,500.
amined. The number of particles was roughly
equal and showed similar variations in size as those shown in Figure 6.
moniatcd mercury ointment, cnn be visualized by electron microscopy in its passage through the cellular layers and intercellular spaces cf the stratum corneum. The sizes of
It was found that with proper processing, mercury, when applied topically either as a electron-dense particles as mercury salts or as
solution of mercuric chlcride or as am- complexes of mercury with protein visible on
ABSORPTION OF MERCURY
327
Fm. 3. This photomicrograph depicts skin treated with mercuric chloride. The area is from middle to lowest layers of the stratum corneum from the same tissue as that depicted
in Figures 1 & 2. In some cell layers electron-dense bodies (x) of unknown nature are present. Stained with uranyl acetate and lead citrate. X43,300.
electron photomierographs were found to vary in glutaraldebyde, treated with ammonium
sulfide and postfixed in osmium tetroxide (Figures 1—5). The use or omission of 1% complexes was obtained when tissue was fixed phosphotungstic acid did not influence the from 50 A to 350 A (see Fig. 1).
The best visualization of mercury or its
328
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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
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e
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Fm. 4. Unstained section of tissue taken from a site of skin to which mercuric chloride had been applied. X30,000.
visualization of the electron-dense particles. to be fewer in number when compared to When specimens of skin from patients that specimens that were dipped in ammonium were treated with a mercurial were not sulfide. This was also true of the tissue fixed dipped in ammonium sulfide some electron- in a mixture of glutaraldebyde and osmium dense particles were present, but they seemed tetroxide or in glutaraldebyde alone. The
ABSORPTION OF MERCURY
FIG.
329
5. Unstained section of stratum corneum from a site where mercuric chloride had
been applied. X50,000.
procedure of carbon coating both sides of the curio sulfide (2). They (P, p) represent either grid tended to minimize migration or subli- mercuric sulfide with or without a surroundmation of mercury in the tissues (7). Only those areas of the skin which had been
ing coat of protein, or mercury particles
which have complexed with tissue proteins, or treated with mercury salts and not the control are particles in aggregation, or combinations
sites treated with the vehicles alone showed of these possibilities. Evidence that they are the presence of the electron-dense particles de- specific precipitates is the following: 1. They scribed above. The unstained sections showed are found only in those sites where mercury the presence of electron-dense particles with salts were applied topically. 2. The electron-
about the same distribution as that of the dense particles are not seen in specimens stained tissue (Figures 4, 5). Occasionally from areas of skin treated with the vehicle osmium tctroxidc fixation resulted in some alone, but processed in an identical manner. black precipitates but, after washing with saline 3. The particles arc seen in unstained as well only minimal amounts were present. as stained tissue. Staining was used to in-
crease contrast, thereby facilitating precise localization of the particles as well as faciliParticles designated P, p on the accom- tating focusing at higher magnifications. 4. panying photomicrographs appear to be some The particles localize in certain definable form of mercury. The range of sizes of the areas and are not diffusely scattered as though tarticles described is similar to that of mer- smeared over the tissue section. 5. They are DIscussION
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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
330
Ftc. 6. Unstained section of normal human skin which was immersed in mercuric
chloride, washed in distilled water and coated with carbon. Variation in size of mercury particles (P, p) is seen. The particles are randomly distributed. X60,000.
found as small aggregates at the outermost Recently Lagerholm and Frithz (8) desurfaces of the stratum corneum, within or scribed the intercellular distribution of meroverlying some but not all cell layers of the cury compound in the epidermis of patients stratum corneum, at desmosomal junctions and with psoriasis. An increase in electron density in the intercellular substance,
of ribosomes, of ehromatin material and of
331
ABSORPTION OF MERCURY
certain intranuclear particles was found in complexes which have migrated through the cells of psoriatic epidermis to which am- stratum corneum by way of the intercellular moniated mercury ointment had been applied
spaces in a maze-like pattern as well as
in vivo.
through the cell layers.
reach their locations in the tissue sections.
Further work of this nature is in progress in the hope that this system can be used to
Several namely:
themselves,
study percutaneous absorption of mercury in human subjects with allergic eczematous con-
1. During the process of cutting sections the cutting instrument may drag mercury
tact dermatitis due to mercury compounds
within five minutes after application of
REFERENCES
The question arises as to how the particles possibilities
suggest
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2. During the process of fixation of the specimens the particles are moved so that they appear to be lying in or on the stratum
iol., 44: 51, 1959.
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F. A. and Finck, H.: The use of specific lar migration in some layers of the stratum 7. Pepe, antibody in electron microscopy. II. The vis-
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All of the results obtained suggest that there
is specific localization of particles (F, p) in sections of skin to which mercury salts had been applied. The particles represent mercury
ualization of mercury-labeled antibody in the electron microscope. J. Biophys. & Biochem. Cytology, 11:
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linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
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