Ultrastructural Changes in the Kidney Following Chronic Exposure to Low Levels of Halothane Louis W. Chang, PhD, Alden W. Dudley Jr, MD, Young K. Lee, MD and Jordan Katz, MD
Rats were subjected to chronic exposure to low levels of halothane (10 and 500 ppm for 8 and 4 weeks, respectively). The ultrastructural changes in the kidneys were studied. Animals exposed to 10 ppm halothane demonstrated chronic degenerative changes in the proximal convoluted tubules, including proliferation and membranous whirling of the basal infoldings of some epithelial cells and membranous degeneration of the mitochondria to form membranous bodies within the cellular cytoplasm. These pathologic changes were even more extensive and exaggerated in animals exposed to 500 ppm halothane. Fusion of the membranous bodies to form large membranous plaques and coalescence of lysosomes to form irregularly shaped cytoplasmic dense bodies were frequently found. Swelling of the mitochondna and areas of focal cytoplasmic degradation were also observed. Extrusions of large cytoplasmic masses containing mitochondria, clusters of smooth endoplasmic reticulum and ribosomes into the tubular lumen were frequently observed. Accumulation of spherical microparticles within the tubular basement membranes were also a prominent finding. The present investigation clearly indicated that halothane is nephrotoxic and may be considered as an occupational hazard. (Am J Pathol 78:225-242, 1975)
SINCE
ITS INTRODUCTION I-
1956,1 halothane or Fluothanes
(1l,l,,-trifluoro-2,2-chlorobromoethane) has gained considerable popularitv as an anesthetic agent because of its controllable, potent, nonexplosive and nonflammable character. Despite its recognized hepatotoxicitv,2- the toxic effects of halothane on other organ systems are still relatively unknown. It has been claimed that some fluorinated anesthetics, methoxvfluorane in particular,9'6 are nephrotoxic. The nephrotoxicitv is largely attributed to the accumulation of inorganic fluoride in the renal tubules.'l- 15,17-9 Although there are no known reports of high output renal failure in anesthetists, it is interesting that Bruce and Colleagues 20 reported a twofold increase in chronic renal disease as a cause of death among anesthetists in the period from 1957 to 1966 over the period from 1947 to 1956. It was during the latter 10-year period that the fluorinated anesthetic agents were introduced. Halothane must be considered a From the Departments of Pathology and Anesthesiology, University of W'isconsin School of Medicine, Madison, WVisc. Accepted for publication September 17, 1974. Address reprint requests to Dr. Louis WV. Chang, Department of Pathology, University of Wisconsin, School of Medicine, 470 North Charter St, Madison, WVI 53706. 225
226
CHANG ET AL
American Journal of Pathology
possible nephrotoxic agent because it is a fluorinated compound. However, because there was no significant alteration in the renal function of human volunteers who were subjected to short-term exposures to acute doses of methoxyflurane and halothane,2' no histologic or cytologic studies of the kidney were performed. Despite the increasing concern that halothane may constitute an occupational hazard,22-28 no detailed investigation was performed to study the pathologic effects of prolonged exposure to low levels of halothane. The present report represents the first detailed morphologic study of the pathologic changes in the kidneys following chronic exposure to halothane. Pathologic changes in other organ systems will be reported elsewhere. Enzyme assays on tissue fractions are also in progress. Materials and Methods Twenty-four Sprague-Dawley rats of both sexes weighing 250 to 350 g were used in this experiment. The animals were divided into three groups, each group consisting of 8 animals. All the animals were housed in specially designed chambers in the University of Wisconsin Biotron where the environmental conditions could be carefully manipulated. Halothane was introduced into the fresh air supply input via a Draegger vaporizer. The concentration levels of halothane were monitored with gas chromatography. Animals in group I were exposed to 10 ppm halothane, 8 hours/day, 5 days/week for 8 weeks. Animals in group II were exposed to 500 ppm halothane, 8 hours/day, 5 days/week for 4 weeks. The control animals (group III) were housed in halothane-free chambers. The animals were sacrificed by intracardiac perfusion with buffered 2% glutaraldehyde. Tissue samples from the kidneys were obtained for both light and electron microscopy. For light microscopy, the kidneys were further fixed in buffered 10% formalin, dehydrated with graded alcohols and embedded in paraffin. Sections of approximately 3 to 4 p were obtained and stained with hematoxylin and eosin, periodic acid-Schiff (PAS) and our recently described silver method29 for the demonstration of cellular injury. For electron microscopy, tissue samples from the renal cortex 'vere carefully obtained and were further fixed in buffered 4% glutaraldehyde at 4 C for 2 hours. The tissues were then postfixed in buffered 1% osmium tetroxide for 1% hours followed by dehydration with graded alcohol and embedding with Epon. Thin sections were cut with a DuPont diamond knife on an LKB Ultrotome III automatic ultramicrotome and were examined with an RCA EMU-3G electron microscope.
Results Light Microscopy
The extent and localization of cellular injury may be indicated by the silver deposits in our new silver technic.-" Silver deposits were observed in the renal proximal convoluted tubular cells (Figure 1) of all animals exposed to halothane, indicating cellular injury. No significant silver deposit was observed in the glomeruli, loop of Henle, distal convoluted tubules or collecting tubules. The kidney sections from the
Vol. 78, No. 2 February 1975
KIDNEY CHANGES AFTER HALOTHANE
---
227
control animals also showed no focal or localizing silver deposit (Figure 2). Both hematoxvlin and eosin and PAS staining demonstrated exfoliation of epithelial cells with the formation of cellular casts in the lumen of the proximal convoluted tubules (Figure 3). These changes were most prominent in animals exposed to 500 ppm halothane; however, they wvere also observed in the 10 ppm subjects. Electron Microscopy
The ultrastructural changes in the kidneys of the rats after exposure to 10 ppm halothane were not very remarkable as compared to normal kidnev (Figure 4). Occasional membranous swirling of the basal infoldings was observed in the proximal convoluted tubules (Figure 5). Membranous bodies, presumably derived from degenerated mitochondria, were also observed in some proximal tubular cells (Figure 6). Pvknotic changes of the tubular cells were onlv an occasional finding. The pathologic changes were much more prominent and frequently observed in animals exposed to 500 ppm halothane. Extensive proliferation and swirling of the basal infoldings was found in many proximal convoluted tubules (Figures 7 and 8). Proliferation of the apical microvilli of these tubular epithelial cells was also occasionally observed. Manv proximal convoluted tubule epithelial cells also demonstrated an accumulation of membranous bodies (Figure 9). Some of these membranous bodies became increasinglv electron dense (Figure 9); others appeared to coalesce, forming large areas of membranous plaque wvithin the cell (Figures 10 and 11). Extrusion of the membranous material into the tubular lumen was frequently observed (Figures 11 and 12). An increase in lysosomes was also prominent in many proximal convoluted tubule cells (Figure 12). Fusion of the Ivsosomes to form irregularly shaped dense bodies (Figures 13 and 14) was observed frequentlv. Vacuolation and accumulation of these cvtoplasmic dense bodies could be observed within manv proximal convoluted tubule cells (Figures 15 and 16). Clusters of smooth endoplasmic reticulum (Figures 12 and 15), areas of focal cytoplasmic degradation (Figure 14), and swelling of the tubular mitochondria (Figures 11 and 16) were occasionally found in the proximal convoluted tubule cells. Extrusion of the Iysosomes. cellular debris and large packages of cvtoplasmic material (Figure 17) into the tubular lumen wvere common findings. Many organelles (including initochondria) within the extruded cytoplasmic packages appeared to be still morphologically intact (Fig-
228
CHANG ET AL
American Journal of Pathology
ures 17 and 18). Large clusters or aggregates of smooth endoplasmic reticulum were also found within the extruded cvtoplasmic masses (Figures 18 and 19). Thickening of the basement membrane was observed in some proximal convoluted tubules. Focal accumulation of spherical microparticles was observed within basement membranes (Figure 20). The spherical microparticles were found to be mainly membranous material and spherical particles of various sizes, presumably cellular debris (Figures 21 and 22). Degenerative changes of the interstitial cells (Figure 22) were occasionally observed. As indicated by light microscopy, no specific pathologic or ultrastructural changes were observed in the glomeruli or distal convoluted tubules. Discussion
Although the toxicity of halothane towards the liver is well recognized,27 the precise pathogenetic mechanism for the halothane hepatitis is still debated.4 Despite the fact that there have been reports concerning the nephrotoxicity of methoxyflurane, a fluorinated anesthetic, the present investigation represents the first detailed report on the nephrotoxicity of halothane. The major metabolic products of halothane are trifluoracetic acid and inorganic bromide.30 Unlike methoxyfluorane only a very minute amount of inorganic fluoride is released from halothane.213031 Therefore, the nephrotoxic effect of halothane is unlikely due to fluoride toxicity, as suggested for methoxyfluorane nephropathy.1315,17-'9 It is possible that, as in the case of some other volatile anesthetics such as chloroform or some other halogenated compounds such as carbon tetrachloride, there may be a toxic intermediate which accumulates within the biologic tissues.30 Cohen and Hood 32'33 revealed an accumulation of metabolites from volatile anesthetics including halothane. However, these metabolites were bound to the cell constituents and were not extractable. From the present investigation, it is obvious that halothane is nephrotoxic and that it affects mainly the biologic membrane system and organelles: plasma membrane (basal infoldings and microvilli), mitochondria, lysosomes and endoplasmic reticulum. The precise pathologic significance of the changes of the basal infoldings is still uncertain. Since halothane alters the transport system of cell membranes,34 it can be speculated that the proliferation and whirling of the basal infoldings may represent an increase in the surface area of the basal plasma mem-
Vol. 78, No. 2 February 1975
KIDNEY CHANGES AFTER HALOTHANE
229
branes for the enhancement of the membrane transport of the PCT cells. Studies on the changes in the renal function and enzvme svstems are in progress. Hopefullyv, these studies will further elucidate the effects of halothane on the renal svstem. The uncoupling effect of halothane on the mitochondria has been well documented.5336 The rapid degeneration of the mitochondria presumablyN gives rise to the large numbers of membranous bodies in the renal tubular cells. MIitochondrial swelling was also occasionally observed in the degenerating tubular cells. Similar mitochondrial swelling w-as observed in animals exposed to methoxNfluorane.'3 WA-e believe that such mitochondrial sw-elling may represent mitochondrial uncoupling and nonspecific changes in cellular degeneration. Accumulation and fuision of lvsosomes to form irregularly shaped dense bodies was observed in many proximal convoluted tubule cells. Similar dense bodies have also been described in methoxvfluorane nephropathb.`3 Enlargement and coalescence of lvsosomes and c-toplasmic bodies wvere also observed in kidney- tubules of choline-deficient rats,3 manganese-deficient mice,-' and in mice with the Chediak-Higashi syndrome.39 In the present in-estigation, conclusions cannot be drawn on the significance and functional nature of these atypical lsosomes. Howev er, it wvas demonstrated that these abnormal structures might be directly related to an alteration in the normal lvsosome function.39 Formation of clusters of smooth endoplasmic reticulum (SER) in the renal tubular cells has also been reported in other toxic conditions." It is believed that the aggregation of SER represents the detoxification response of the kidney. Extrusion of such SER aggregations in cvtoplasmic masses into the tubular lumen has also been described.40 These SER aggregates probably represent the hvperplastic, hvpoactive ER and could account for their selective extrusion in cvtoplasmic masses throu,gh a process of potocvtosis.42q4 Focal cvtoplasmic degradation was originally described in liv-er cells after intoxication bv various compounds.45 These 'were focal degenerative areas containing cvtoplasmic debris and were considered to be a special form of cellular degeneration after injury. The focal cvtoplasmic degradation observed within the proximal convoluted tubule epithelial cells after halothane intoxication probably represents this phenomenon. Similar focal cvtoplasmic degradation lesions have been described in nerve cells 46 and in hepatocv'tes 4 4- following methbyl mercurv- intoxication. The occurrence of clusters of spherical microparticles within the basement membrane has recently- been described by Burkholder and co-
230
CHANG ET AL
American Journal of Pathology
workers 49 in renal glomerular diseases. The particles had an average size of 500 to 580 A and were pleomorphic but typically had a dense core with a limiting membrane. Some oval, ruptured or giant forms were seen, and frequently many particles had clear cores. It is believed that the majority of these particles may represent microvesicles discharged from cells during cellular injury.49 Our present observation represents the first report on the association of spherical membrane particles with renal tubular injury. The present investigation certainly indicates that halothane is not only hepatotoxic 2-7 and neurotoxic,5" but also nephrotoxic. Moreover, since the present study was designed to simulate the actual conditions of an operating room (the concentration of halothane in the ambient air of the operating room ranges from 1 to 500 ppm, depending on the sampling sites) and the average working conditions of operating room personnel (8 hours/day and 5 days/week), the results from the present investigation strongly supports the notion that halothane may be considered to be an occupational hazard. References 1. Raventos J: Action of fluothane: a new volatile anesthetic. Br J Pharmacol 11:394-398, 1956 2. Qizilbash AH: Halothane hepatitis. Can Med Assoc J 108:171-177, 1973 3. Ngai SH: Hepatic effects of halothane: reviews of the literature, The National Halothane Study. Edited bv Bunker JP, Forrest WH, Mosteller F, Vandam LD. Bethesda, Md, National Institutes of Health and National Institute of General Medical Sciences, 1969, pp 11-18 4. Carney FMT, Van Dyke RA:Halothane hepatitis: a critical review. Anesth
Analg (Cleve) 51:135-160, 1972 5. Ross WT Jr, Cardell RR: Effects of halothane on the ultrastructlure of rat liver cells. Am J Anat 135:5-22, 1972 6. Hughes HC, Lang CM: Hepatic necrosis produced by repeated administration of halothane to guinea pigs. Anesthesiology 36:466-471, 1972 7. Chang LW, Dudley AW Jr, Katz J: Ultrastructural evidence of hepatic injuries induced by chronic exposure to low levels of halothane. Am J Pathol 74:103a-104a, 1974 (abstr) 8. Crandell WB, Pappas SG, MacDonald A: Nephrotoxicity associated with methoxyfluorane anesthesia. Anesthesiology 27:591-607, 1966 9. Pezzi PJ, Frobese AS, Greenberg SR: Methoxyflurane and renal toxicity. Lancet 1:823, 1966 10. Elkington SG, Goffinet JA, Conn HO: Renal and hepatic injury associated with methoxyflurane anesthesia. Ann Intern Med 69:1229-1236, 1968 11. Kuzucu EY: Methoxyflurane, tetracycline and renal failure. JAMA 211: 1162-1164, 1970 12. Dobkin AB: Methoxyflurane and renal function (Letter to the editor). JAMA 219:750, 1972 13. Kosek, JC, Mazze RI, Cousins MJ: The morphology and pathogenesis of
Vol. 78, No. 2 February 1975
14. 15. 16.
17. 18. 19. 20.
21. 22.
23. 24.
25.
26. 27.
28.
29. 30. 31. 32. 33.
KIDNEY CHANGES AFTER HALOTHANE
231
nephrotoxicitv following methoxvflurane (penthrane) anesthesia: an experimental model in rats. Lab Invest 27:575-580, 1972 Hetrick WVD, Wolfson B, Garcia DA, Siker ES: Renal responses to light methoxyflurane anesthesia. Anesthesiology 38:30-37, 1973 Mazze RI, Cousins RJ: Renal toxicity of anesthetics: with specific references to the nephrotoxicity of methoxvflurane. Can Anaesth Soc J 20:64-80, 1973 Cousins MJ, Mazze RI, Barr GA, Kosek JC: A comparison of the renal effects of isoflurane and methoxyflurane in Fischer 344 rats. Anesthesiology 38:557-563, 1973 Taves DR, Fry BW, Freeman RB: Toxicity following methoxvflurane anesthesia. II. Fluoride concentration in nephrotoxicity. JAMA 214:91-95, 1970 Mazze RI, Trudell JR, Cousins MJ: Methoxyflurane metabolism and renal dysfunction: clinical correlation in man. Anesthesiology 35:247-252, 1971 Mlazze RI, Cousins MJ, Kosek JC: Dose-related methoxvflurane nephrotoxicitv in rats: a biochemical and pathologic correlation. Anesthesiology 36:571-587, 1972 Bruce DL, Eide KA, Linde HW, Eckenhoff JE: Causes of death among anesthesiologists: a 20 year survey. Anesthesiology 29:565-569, 1968. Tobey RE, Clubb RJ: Renal function after methoxvflurane and halothane anesthesia. JAMA 223:649-652, 1973 Linde HW, Bruce DL: Occupational exposure of anesthetists to halothane, nitrous oxide and radiation. Anesthesiology 30:363-386, 1969 Whitcher CE, Cohen EN, Trudell JR: Chronic exposure to anesthetic gases in the operating room. Anesthesiology 35:348-356, 1971 Corbett TH: Anesthetics as a cause of abortion. Fertil Steril 23:866-869, 1972 Jenkins LC: Chronic exposure to anesthetics: a toxicity problem? Can Anaesth Soc J 20:104-120, 1973 Corbett TH:Retention of anesthetic agents following occupational exposure. Anesth Analg (Cleve) 52:614-618, 1973 ASA Scientific Panel: A preliminary report: the hazard of trace anesthetics in unvented operating rooms. Anesth Rev 1:22-23, 1974 Chenoweth MB, Leong BKJ, Sparchu GL, Torkelson TR: Toxicities of methoxyflurane, halothane and diethyl ether in laboratory animals on rerepeated inhalation at subanesthetic concentrations. Cellular Biology and Toxicity of Anesthesia. Edited by BR Fink. Baltimore, The Williams and Wilis Co, 1972, pp 275-285 Chang LWV, Dudley AW Jr, Potter CE: A silver technique for the study of cellular injuries. Histo-Logic 4:47-48, 1974 Van Dvke RA: Biotransformation of volatile anaesthetics 'with special emphasis on the role of metabolism in the toxicity of anesthetics. Can Anaesth Soc J 20:21-3, 1973 Creasser C, Stoelting RK: Serum inorganic fluoride concentrations during and after halothane, fluroxene and methoxvflurane anesthesia in man. Anesthesiology 39:537-540, 1973 Cohen EN, Hood N: Application of low-temperature autoradiography to studies of the uptake and metabolism of volatile anesthetics in the mouse. I. Chloroform. Anesthesiology 30:306-314, 1969 Cohen EN, Hood N: Application of low-temperature autoradiography to
232
34.
35. 36.
37. 38. 39. 40.
41. 42. 43.
44.
45. 46.
47. 48.
49. 50.
CHANG ET AL
American Journal of Pathology
studies of the uptake and metabolism of volatile anesthetics in the mouse. II. Diethyl ether. Anesthesiology 31:61-68, 1969 Andersen NB, Amaranath L: Anesthetic effects on transport across cell membranes. Anesthesiology 39:126-152, 1973 Taylor, CA, Williams CH, Wakabayashi T, Valdivia E, Harris RA, Green DE: The effect of halothane on energized configurational changes in heart mitochondria in situ.28 pp 117-127 Cohen PJ: Effect of anesthetics on mitochondrial function. Anesthesiology 39:153-164, 1973 Newberne PM, Bresnahan MR, Kula N: Effects of two synthetic antioxidants, vitamin E and ascorbic acid on the choline-deficient rat. J Nutr 97:219-227, 1969 Bell LT, Hurley LS: Ultrastructural effects of manganese deficiency in liver, heart, kidney, and pancreas of mice. Lab Invest 29:723-736, 1973 Prieur DJ, Davis WC, Padgett GA: Defective function of renal lysosomes in mice with the Chediak-Higashi syndrome. Am J Pathol 67:227-236, 1972 Fowler BA:The morphologic effects of dieldrin and methyl mercuric chloride on pars recta segments of rat kidney proximal tubules. Am J Pathol 69:163178, 1972 Fowler BA:Ultrastructural evidence for nephropathy induced by long-term exposure to small amounts of methyl mercury. Science 175:780-781, 1972 Ericsson JLZ, Trump BF: Electron microscopy of the uriniferous tubules, The Kidney, Vol 1. Edited by C Rouiller, AF Muller. New York, Academic Press, Inc, 1969, pp 351-447 Chatelanat F, Simon GT: Ultrastructural pathology of the tubules and interstitial tissue.42 pp 351-447 Zollinger HU: Cytologic studies with the phase microscope. I. The formation of "blisters" on cells in suspension (potocytosis), with observations on the nature of the cellular membrane. Am J Pathol 24:545-567, 1948 Hruban Z, Spargo B, Swift H, Wissler RW, Kleinfeld RG: Focal cytoplasmic degradation. Am J Pathol 42:657-683, 1963 Chang LW, Hartmann HA: Ultrastructural studies of the nervous system after mercury intoxication. I. Pathological changes in the nerve cell bodies. Acta Neuropathol 20:122-138, 1972 Chang LW, Yamaguchi S: Ultrastructural changes in the liver following long term diet of mercury contaminated tuna. Environ Res 7:133-148, 1974 Desnoyers PA, Chang LW: Ultrastructural changes in rat hepatocytes following acute methyl mercury intoxication. Environ Res (In press), 1975 Burkholder PM, Hyman LR, Barber TA: Extracellular clusters of spherical microparticles in glomeruli in human renal glomerular diseases. Lab Invest 28:415-425, 1973 Chang LW, Dudley AW Jr, Lee YK, Katz J: Ultrastructural changes in the nervous system after chronic exposure to halothane. Exp Neurol 45: 209-219, 1974
Acknowledgments The authors wish to thank Drs. P. M. Burkholder, J. Allen and D. Slautterback of the Departments of Pathology and Anatomy for their kind opinions and consultation throughout the work.
gw =S;; -~~,
5,
M
t
2
1 ~~~ U
~
~
4
O's.
fe-/
v
3
Fig 1-Kidney -from rat given 500 ppm halothane. Note the selective silver deposit in of the proximal convoluted tubules, indicating cellular injury in these tubules (Silver Fig 2-Control rat kidney. No significant silver deposit was observed stain, x 150). Fig 3-Kidney from rat given 500 ppm halothane. Exfoliation (Silver stain, x 150). of epithelial cells (arrows) and cellular casts within the lumen of the proximal convoluted tubules (PCT); the collecting duct (CD) appeared to be normal (PAS, X 650). some
9A
'.t 5
e
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Ai%
. j
Fig 4Proximal convoluted tubule (POT) from normal rat. MV=microvilli, BM= basement membrane (X 12,000). Fig 5PCT from rat treated with 10 ppm halothane. Note the membranous swirling of the basal infoldings (arrows) at the base of the epithelial cell. Fig 6PCT from rat given 10 ppm N-nucleus, BM= basement membrane (X 12,000). halothane. Degradation of mitochondria to form large membranous bodies (MB). Remnant of a mitochondria (arrow) could still be recognized (X 17,000).
F
10~ ~
W
~~~~
4--
-%~~~~~~~~~~k Fig 7-PCT from rat treated with 500 ppm halothane. Severe swirling of the basal infoldings. BM=basement membrane, M=mitochondria (x 17,000). Fig 8-PCT from rat administered 500 ppm halothane. Extensive proliferation of the basal infoldings. Displacement of the mitochondria to the apical portion of the cells is also evident. BM=basement membrane (x 12,000).
4k
f
40
'4'~~~~4
J~~~~~~~~~l~~~l
10
-,-w-~~A-
Fig 9-PCT from rat administered 500 ppm halothane. Accumulation of membranous bodies was observed. Many of these bodies were associated with electron-dense particles and became increasingly electron dense. N=nucleus, BM=basement membrane (x 17,000). Fig 10-PCT from rat given 500 ppm halothane. Coalescence of the membranous bodies formed large areas of membranous plaques (MP) within the cell. MV=microvilli (X 17,000).
-@-~~~~~~~~~~~~.
1-41
Fig 11-PCT from rat given 500 ppm halothane. Large areas of membranous plaque (MJP) were seen within the cell; extrusion of the membranous materials into the tubular lumen (TL) was also evident. Large swollen mitochondria in a neighboring cells were also observed. MV-microvilli, N=nucleus (X 12,000). Fig 12-T from rat administered
500 ppm halothane. Extruded membranous materials were seen within the tubular lumen. Increased numbers of Iysosomes and clusters of smooth endoplasmic reticulum (arrow) were also obsenrved. MUV-microvrilli, N=nucleus (X 12,000).
|§W0rlSmq^at.#-:;e5sS$i.,>tl{-X:as@¢.e|84,R1i_7.Sr!~ ~FiqW,BS
.;
L@. .si-t
ii_ VM.eeflq3
._
*K~~~~~~~~~~~~~~~~~~~~~~~~~~~~Win.,s
Xe4it/Sk.t'
2
W
40
*r1r_,xU )= 4z.t} ¢ s^;W,X
':'O'
t'rwe
i$ w 6i;5|F-2.iP .Ae:i.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ s_'s-,, X ,t
"*:B :e F _.
r
f
.s r,
il. ,..:w.
~
;g
tae
A
lage ireual
shpe
(pesmal degrade proein
des
were
~
;_
ta
blody(B
also obere
4 >; It; | ts
s>wasfsfon x 12000_
withi
t - wf Ji>
the
* M je ii,V
epiheia
cl. _
'"
.s'
2.~~~~~~~~~~~~~~~~~~~~~4 4P.~~~
r
~ ~
~
-=
_
t.w..,t,
Fig 15-PCT from rat administered 500 ppm halothane. Accumulation of cytoplasmic dense bodies within a tubular epithelial cell. Many of these cytoplasmic dense bodies appeared to be vacuolated. A large cluster of smooth endoplasmic reticulum (SER) was also observed. N=nucleus (x 26,000). Fig 16-PCT from rat administered 500 ppm halothane. A pyknotic epithelial cell, which became very electron dense, contained many cytoplasmic dense bodies and large swollen mitochondria (x 12,000).
1 7~~.
_iR,wt.
I'D^
,S .=n
.vo
o
.:.S;;^.
_ 7.A
Fig 17-Extrusion of cellular debris, lysosomes and packages of cytoplasmic masses containing mitochondria and other organelles were found within the tubular lumen of many proximal convoluted tubules. MV=microvilli, N=nucleus (X 7750). Fig 18A large package of cytoplasmic mass containing clusters of smooth endoplasmic reticulum (SER), free polyribosomes, and mitochondria was found within the tubular lumen. The microvilli (MV) of the tubules were being compressed by the cytoplasmic mass. N= nucleus (X 17,000). Fig 19-Large cytoplasmic masses within the tubular lumen. A large cluster of smooth endoplasmic reticulum (SER) was included in a cytoplasmic mass. (X 12,000).
2
e20 ;
Rats were subjected to chronic exposure to low levels of halothane (10 and 500 ppm for 8 and 4 weeks, respectively). The ultrastructural changes in the kidneys were studied. Animals exposed to 10 ppm halothane demonstrated chronic degenerative changes in the proximal convoluted tubules, including proliferation and membranous whirling of the basal infoldings of some epithelial cells and membranous degeneration of the mitochondria to form membranous bodies within the cellular cytoplasm. These pathologic changes were even more extensive and exaggerated in animals exposed to 500 ppm halothane. Fusion of the membranous bodies to form large membranous plaques and coalescence of lysosomes to form irregularly shaped cytoplasmic dense bodies were frequently found. Swelling of the mitochondna and areas of focal cytoplasmic degradation were also observed. Extrusions of large cytoplasmic masses containing mitochondria, clusters of smooth endoplasmic reticulum and ribosomes into the tubular lumen were frequently observed. Accumulation of spherical microparticles within the tubular basement membranes were also a prominent finding. The present investigation clearly indicated that halothane is nephrotoxic and may be considered as an occupational hazard. (Am J Pathol 78:225-242, 1975)
SINCE
ITS INTRODUCTION I-
1956,1 halothane or Fluothanes
(1l,l,,-trifluoro-2,2-chlorobromoethane) has gained considerable popularitv as an anesthetic agent because of its controllable, potent, nonexplosive and nonflammable character. Despite its recognized hepatotoxicitv,2- the toxic effects of halothane on other organ systems are still relatively unknown. It has been claimed that some fluorinated anesthetics, methoxvfluorane in particular,9'6 are nephrotoxic. The nephrotoxicitv is largely attributed to the accumulation of inorganic fluoride in the renal tubules.'l- 15,17-9 Although there are no known reports of high output renal failure in anesthetists, it is interesting that Bruce and Colleagues 20 reported a twofold increase in chronic renal disease as a cause of death among anesthetists in the period from 1957 to 1966 over the period from 1947 to 1956. It was during the latter 10-year period that the fluorinated anesthetic agents were introduced. Halothane must be considered a From the Departments of Pathology and Anesthesiology, University of W'isconsin School of Medicine, Madison, WVisc. Accepted for publication September 17, 1974. Address reprint requests to Dr. Louis WV. Chang, Department of Pathology, University of Wisconsin, School of Medicine, 470 North Charter St, Madison, WVI 53706. 225
226
CHANG ET AL
American Journal of Pathology
possible nephrotoxic agent because it is a fluorinated compound. However, because there was no significant alteration in the renal function of human volunteers who were subjected to short-term exposures to acute doses of methoxyflurane and halothane,2' no histologic or cytologic studies of the kidney were performed. Despite the increasing concern that halothane may constitute an occupational hazard,22-28 no detailed investigation was performed to study the pathologic effects of prolonged exposure to low levels of halothane. The present report represents the first detailed morphologic study of the pathologic changes in the kidneys following chronic exposure to halothane. Pathologic changes in other organ systems will be reported elsewhere. Enzyme assays on tissue fractions are also in progress. Materials and Methods Twenty-four Sprague-Dawley rats of both sexes weighing 250 to 350 g were used in this experiment. The animals were divided into three groups, each group consisting of 8 animals. All the animals were housed in specially designed chambers in the University of Wisconsin Biotron where the environmental conditions could be carefully manipulated. Halothane was introduced into the fresh air supply input via a Draegger vaporizer. The concentration levels of halothane were monitored with gas chromatography. Animals in group I were exposed to 10 ppm halothane, 8 hours/day, 5 days/week for 8 weeks. Animals in group II were exposed to 500 ppm halothane, 8 hours/day, 5 days/week for 4 weeks. The control animals (group III) were housed in halothane-free chambers. The animals were sacrificed by intracardiac perfusion with buffered 2% glutaraldehyde. Tissue samples from the kidneys were obtained for both light and electron microscopy. For light microscopy, the kidneys were further fixed in buffered 10% formalin, dehydrated with graded alcohols and embedded in paraffin. Sections of approximately 3 to 4 p were obtained and stained with hematoxylin and eosin, periodic acid-Schiff (PAS) and our recently described silver method29 for the demonstration of cellular injury. For electron microscopy, tissue samples from the renal cortex 'vere carefully obtained and were further fixed in buffered 4% glutaraldehyde at 4 C for 2 hours. The tissues were then postfixed in buffered 1% osmium tetroxide for 1% hours followed by dehydration with graded alcohol and embedding with Epon. Thin sections were cut with a DuPont diamond knife on an LKB Ultrotome III automatic ultramicrotome and were examined with an RCA EMU-3G electron microscope.
Results Light Microscopy
The extent and localization of cellular injury may be indicated by the silver deposits in our new silver technic.-" Silver deposits were observed in the renal proximal convoluted tubular cells (Figure 1) of all animals exposed to halothane, indicating cellular injury. No significant silver deposit was observed in the glomeruli, loop of Henle, distal convoluted tubules or collecting tubules. The kidney sections from the
Vol. 78, No. 2 February 1975
KIDNEY CHANGES AFTER HALOTHANE
---
227
control animals also showed no focal or localizing silver deposit (Figure 2). Both hematoxvlin and eosin and PAS staining demonstrated exfoliation of epithelial cells with the formation of cellular casts in the lumen of the proximal convoluted tubules (Figure 3). These changes were most prominent in animals exposed to 500 ppm halothane; however, they wvere also observed in the 10 ppm subjects. Electron Microscopy
The ultrastructural changes in the kidneys of the rats after exposure to 10 ppm halothane were not very remarkable as compared to normal kidnev (Figure 4). Occasional membranous swirling of the basal infoldings was observed in the proximal convoluted tubules (Figure 5). Membranous bodies, presumably derived from degenerated mitochondria, were also observed in some proximal tubular cells (Figure 6). Pvknotic changes of the tubular cells were onlv an occasional finding. The pathologic changes were much more prominent and frequently observed in animals exposed to 500 ppm halothane. Extensive proliferation and swirling of the basal infoldings was found in many proximal convoluted tubules (Figures 7 and 8). Proliferation of the apical microvilli of these tubular epithelial cells was also occasionally observed. Manv proximal convoluted tubule epithelial cells also demonstrated an accumulation of membranous bodies (Figure 9). Some of these membranous bodies became increasinglv electron dense (Figure 9); others appeared to coalesce, forming large areas of membranous plaque wvithin the cell (Figures 10 and 11). Extrusion of the membranous material into the tubular lumen was frequently observed (Figures 11 and 12). An increase in lysosomes was also prominent in many proximal convoluted tubule cells (Figure 12). Fusion of the Ivsosomes to form irregularly shaped dense bodies (Figures 13 and 14) was observed frequentlv. Vacuolation and accumulation of these cvtoplasmic dense bodies could be observed within manv proximal convoluted tubule cells (Figures 15 and 16). Clusters of smooth endoplasmic reticulum (Figures 12 and 15), areas of focal cytoplasmic degradation (Figure 14), and swelling of the tubular mitochondria (Figures 11 and 16) were occasionally found in the proximal convoluted tubule cells. Extrusion of the Iysosomes. cellular debris and large packages of cvtoplasmic material (Figure 17) into the tubular lumen wvere common findings. Many organelles (including initochondria) within the extruded cytoplasmic packages appeared to be still morphologically intact (Fig-
228
CHANG ET AL
American Journal of Pathology
ures 17 and 18). Large clusters or aggregates of smooth endoplasmic reticulum were also found within the extruded cvtoplasmic masses (Figures 18 and 19). Thickening of the basement membrane was observed in some proximal convoluted tubules. Focal accumulation of spherical microparticles was observed within basement membranes (Figure 20). The spherical microparticles were found to be mainly membranous material and spherical particles of various sizes, presumably cellular debris (Figures 21 and 22). Degenerative changes of the interstitial cells (Figure 22) were occasionally observed. As indicated by light microscopy, no specific pathologic or ultrastructural changes were observed in the glomeruli or distal convoluted tubules. Discussion
Although the toxicity of halothane towards the liver is well recognized,27 the precise pathogenetic mechanism for the halothane hepatitis is still debated.4 Despite the fact that there have been reports concerning the nephrotoxicity of methoxyflurane, a fluorinated anesthetic, the present investigation represents the first detailed report on the nephrotoxicity of halothane. The major metabolic products of halothane are trifluoracetic acid and inorganic bromide.30 Unlike methoxyfluorane only a very minute amount of inorganic fluoride is released from halothane.213031 Therefore, the nephrotoxic effect of halothane is unlikely due to fluoride toxicity, as suggested for methoxyfluorane nephropathy.1315,17-'9 It is possible that, as in the case of some other volatile anesthetics such as chloroform or some other halogenated compounds such as carbon tetrachloride, there may be a toxic intermediate which accumulates within the biologic tissues.30 Cohen and Hood 32'33 revealed an accumulation of metabolites from volatile anesthetics including halothane. However, these metabolites were bound to the cell constituents and were not extractable. From the present investigation, it is obvious that halothane is nephrotoxic and that it affects mainly the biologic membrane system and organelles: plasma membrane (basal infoldings and microvilli), mitochondria, lysosomes and endoplasmic reticulum. The precise pathologic significance of the changes of the basal infoldings is still uncertain. Since halothane alters the transport system of cell membranes,34 it can be speculated that the proliferation and whirling of the basal infoldings may represent an increase in the surface area of the basal plasma mem-
Vol. 78, No. 2 February 1975
KIDNEY CHANGES AFTER HALOTHANE
229
branes for the enhancement of the membrane transport of the PCT cells. Studies on the changes in the renal function and enzvme svstems are in progress. Hopefullyv, these studies will further elucidate the effects of halothane on the renal svstem. The uncoupling effect of halothane on the mitochondria has been well documented.5336 The rapid degeneration of the mitochondria presumablyN gives rise to the large numbers of membranous bodies in the renal tubular cells. MIitochondrial swelling was also occasionally observed in the degenerating tubular cells. Similar mitochondrial swelling w-as observed in animals exposed to methoxNfluorane.'3 WA-e believe that such mitochondrial sw-elling may represent mitochondrial uncoupling and nonspecific changes in cellular degeneration. Accumulation and fuision of lvsosomes to form irregularly shaped dense bodies was observed in many proximal convoluted tubule cells. Similar dense bodies have also been described in methoxvfluorane nephropathb.`3 Enlargement and coalescence of lvsosomes and c-toplasmic bodies wvere also observed in kidney- tubules of choline-deficient rats,3 manganese-deficient mice,-' and in mice with the Chediak-Higashi syndrome.39 In the present in-estigation, conclusions cannot be drawn on the significance and functional nature of these atypical lsosomes. Howev er, it wvas demonstrated that these abnormal structures might be directly related to an alteration in the normal lvsosome function.39 Formation of clusters of smooth endoplasmic reticulum (SER) in the renal tubular cells has also been reported in other toxic conditions." It is believed that the aggregation of SER represents the detoxification response of the kidney. Extrusion of such SER aggregations in cvtoplasmic masses into the tubular lumen has also been described.40 These SER aggregates probably represent the hvperplastic, hvpoactive ER and could account for their selective extrusion in cvtoplasmic masses throu,gh a process of potocvtosis.42q4 Focal cvtoplasmic degradation was originally described in liv-er cells after intoxication bv various compounds.45 These 'were focal degenerative areas containing cvtoplasmic debris and were considered to be a special form of cellular degeneration after injury. The focal cvtoplasmic degradation observed within the proximal convoluted tubule epithelial cells after halothane intoxication probably represents this phenomenon. Similar focal cvtoplasmic degradation lesions have been described in nerve cells 46 and in hepatocv'tes 4 4- following methbyl mercurv- intoxication. The occurrence of clusters of spherical microparticles within the basement membrane has recently- been described by Burkholder and co-
230
CHANG ET AL
American Journal of Pathology
workers 49 in renal glomerular diseases. The particles had an average size of 500 to 580 A and were pleomorphic but typically had a dense core with a limiting membrane. Some oval, ruptured or giant forms were seen, and frequently many particles had clear cores. It is believed that the majority of these particles may represent microvesicles discharged from cells during cellular injury.49 Our present observation represents the first report on the association of spherical membrane particles with renal tubular injury. The present investigation certainly indicates that halothane is not only hepatotoxic 2-7 and neurotoxic,5" but also nephrotoxic. Moreover, since the present study was designed to simulate the actual conditions of an operating room (the concentration of halothane in the ambient air of the operating room ranges from 1 to 500 ppm, depending on the sampling sites) and the average working conditions of operating room personnel (8 hours/day and 5 days/week), the results from the present investigation strongly supports the notion that halothane may be considered to be an occupational hazard. References 1. Raventos J: Action of fluothane: a new volatile anesthetic. Br J Pharmacol 11:394-398, 1956 2. Qizilbash AH: Halothane hepatitis. Can Med Assoc J 108:171-177, 1973 3. Ngai SH: Hepatic effects of halothane: reviews of the literature, The National Halothane Study. Edited bv Bunker JP, Forrest WH, Mosteller F, Vandam LD. Bethesda, Md, National Institutes of Health and National Institute of General Medical Sciences, 1969, pp 11-18 4. Carney FMT, Van Dyke RA:Halothane hepatitis: a critical review. Anesth
Analg (Cleve) 51:135-160, 1972 5. Ross WT Jr, Cardell RR: Effects of halothane on the ultrastructlure of rat liver cells. Am J Anat 135:5-22, 1972 6. Hughes HC, Lang CM: Hepatic necrosis produced by repeated administration of halothane to guinea pigs. Anesthesiology 36:466-471, 1972 7. Chang LW, Dudley AW Jr, Katz J: Ultrastructural evidence of hepatic injuries induced by chronic exposure to low levels of halothane. Am J Pathol 74:103a-104a, 1974 (abstr) 8. Crandell WB, Pappas SG, MacDonald A: Nephrotoxicity associated with methoxyfluorane anesthesia. Anesthesiology 27:591-607, 1966 9. Pezzi PJ, Frobese AS, Greenberg SR: Methoxyflurane and renal toxicity. Lancet 1:823, 1966 10. Elkington SG, Goffinet JA, Conn HO: Renal and hepatic injury associated with methoxyflurane anesthesia. Ann Intern Med 69:1229-1236, 1968 11. Kuzucu EY: Methoxyflurane, tetracycline and renal failure. JAMA 211: 1162-1164, 1970 12. Dobkin AB: Methoxyflurane and renal function (Letter to the editor). JAMA 219:750, 1972 13. Kosek, JC, Mazze RI, Cousins MJ: The morphology and pathogenesis of
Vol. 78, No. 2 February 1975
14. 15. 16.
17. 18. 19. 20.
21. 22.
23. 24.
25.
26. 27.
28.
29. 30. 31. 32. 33.
KIDNEY CHANGES AFTER HALOTHANE
231
nephrotoxicitv following methoxvflurane (penthrane) anesthesia: an experimental model in rats. Lab Invest 27:575-580, 1972 Hetrick WVD, Wolfson B, Garcia DA, Siker ES: Renal responses to light methoxyflurane anesthesia. Anesthesiology 38:30-37, 1973 Mazze RI, Cousins RJ: Renal toxicity of anesthetics: with specific references to the nephrotoxicity of methoxvflurane. Can Anaesth Soc J 20:64-80, 1973 Cousins MJ, Mazze RI, Barr GA, Kosek JC: A comparison of the renal effects of isoflurane and methoxyflurane in Fischer 344 rats. Anesthesiology 38:557-563, 1973 Taves DR, Fry BW, Freeman RB: Toxicity following methoxvflurane anesthesia. II. Fluoride concentration in nephrotoxicity. JAMA 214:91-95, 1970 Mazze RI, Trudell JR, Cousins MJ: Methoxyflurane metabolism and renal dysfunction: clinical correlation in man. Anesthesiology 35:247-252, 1971 Mlazze RI, Cousins MJ, Kosek JC: Dose-related methoxvflurane nephrotoxicitv in rats: a biochemical and pathologic correlation. Anesthesiology 36:571-587, 1972 Bruce DL, Eide KA, Linde HW, Eckenhoff JE: Causes of death among anesthesiologists: a 20 year survey. Anesthesiology 29:565-569, 1968. Tobey RE, Clubb RJ: Renal function after methoxvflurane and halothane anesthesia. JAMA 223:649-652, 1973 Linde HW, Bruce DL: Occupational exposure of anesthetists to halothane, nitrous oxide and radiation. Anesthesiology 30:363-386, 1969 Whitcher CE, Cohen EN, Trudell JR: Chronic exposure to anesthetic gases in the operating room. Anesthesiology 35:348-356, 1971 Corbett TH: Anesthetics as a cause of abortion. Fertil Steril 23:866-869, 1972 Jenkins LC: Chronic exposure to anesthetics: a toxicity problem? Can Anaesth Soc J 20:104-120, 1973 Corbett TH:Retention of anesthetic agents following occupational exposure. Anesth Analg (Cleve) 52:614-618, 1973 ASA Scientific Panel: A preliminary report: the hazard of trace anesthetics in unvented operating rooms. Anesth Rev 1:22-23, 1974 Chenoweth MB, Leong BKJ, Sparchu GL, Torkelson TR: Toxicities of methoxyflurane, halothane and diethyl ether in laboratory animals on rerepeated inhalation at subanesthetic concentrations. Cellular Biology and Toxicity of Anesthesia. Edited by BR Fink. Baltimore, The Williams and Wilis Co, 1972, pp 275-285 Chang LWV, Dudley AW Jr, Potter CE: A silver technique for the study of cellular injuries. Histo-Logic 4:47-48, 1974 Van Dvke RA: Biotransformation of volatile anaesthetics 'with special emphasis on the role of metabolism in the toxicity of anesthetics. Can Anaesth Soc J 20:21-3, 1973 Creasser C, Stoelting RK: Serum inorganic fluoride concentrations during and after halothane, fluroxene and methoxvflurane anesthesia in man. Anesthesiology 39:537-540, 1973 Cohen EN, Hood N: Application of low-temperature autoradiography to studies of the uptake and metabolism of volatile anesthetics in the mouse. I. Chloroform. Anesthesiology 30:306-314, 1969 Cohen EN, Hood N: Application of low-temperature autoradiography to
232
34.
35. 36.
37. 38. 39. 40.
41. 42. 43.
44.
45. 46.
47. 48.
49. 50.
CHANG ET AL
American Journal of Pathology
studies of the uptake and metabolism of volatile anesthetics in the mouse. II. Diethyl ether. Anesthesiology 31:61-68, 1969 Andersen NB, Amaranath L: Anesthetic effects on transport across cell membranes. Anesthesiology 39:126-152, 1973 Taylor, CA, Williams CH, Wakabayashi T, Valdivia E, Harris RA, Green DE: The effect of halothane on energized configurational changes in heart mitochondria in situ.28 pp 117-127 Cohen PJ: Effect of anesthetics on mitochondrial function. Anesthesiology 39:153-164, 1973 Newberne PM, Bresnahan MR, Kula N: Effects of two synthetic antioxidants, vitamin E and ascorbic acid on the choline-deficient rat. J Nutr 97:219-227, 1969 Bell LT, Hurley LS: Ultrastructural effects of manganese deficiency in liver, heart, kidney, and pancreas of mice. Lab Invest 29:723-736, 1973 Prieur DJ, Davis WC, Padgett GA: Defective function of renal lysosomes in mice with the Chediak-Higashi syndrome. Am J Pathol 67:227-236, 1972 Fowler BA:The morphologic effects of dieldrin and methyl mercuric chloride on pars recta segments of rat kidney proximal tubules. Am J Pathol 69:163178, 1972 Fowler BA:Ultrastructural evidence for nephropathy induced by long-term exposure to small amounts of methyl mercury. Science 175:780-781, 1972 Ericsson JLZ, Trump BF: Electron microscopy of the uriniferous tubules, The Kidney, Vol 1. Edited by C Rouiller, AF Muller. New York, Academic Press, Inc, 1969, pp 351-447 Chatelanat F, Simon GT: Ultrastructural pathology of the tubules and interstitial tissue.42 pp 351-447 Zollinger HU: Cytologic studies with the phase microscope. I. The formation of "blisters" on cells in suspension (potocytosis), with observations on the nature of the cellular membrane. Am J Pathol 24:545-567, 1948 Hruban Z, Spargo B, Swift H, Wissler RW, Kleinfeld RG: Focal cytoplasmic degradation. Am J Pathol 42:657-683, 1963 Chang LW, Hartmann HA: Ultrastructural studies of the nervous system after mercury intoxication. I. Pathological changes in the nerve cell bodies. Acta Neuropathol 20:122-138, 1972 Chang LW, Yamaguchi S: Ultrastructural changes in the liver following long term diet of mercury contaminated tuna. Environ Res 7:133-148, 1974 Desnoyers PA, Chang LW: Ultrastructural changes in rat hepatocytes following acute methyl mercury intoxication. Environ Res (In press), 1975 Burkholder PM, Hyman LR, Barber TA: Extracellular clusters of spherical microparticles in glomeruli in human renal glomerular diseases. Lab Invest 28:415-425, 1973 Chang LW, Dudley AW Jr, Lee YK, Katz J: Ultrastructural changes in the nervous system after chronic exposure to halothane. Exp Neurol 45: 209-219, 1974
Acknowledgments The authors wish to thank Drs. P. M. Burkholder, J. Allen and D. Slautterback of the Departments of Pathology and Anatomy for their kind opinions and consultation throughout the work.
gw =S;; -~~,
5,
M
t
2
1 ~~~ U
~
~
4
O's.
fe-/
v
3
Fig 1-Kidney -from rat given 500 ppm halothane. Note the selective silver deposit in of the proximal convoluted tubules, indicating cellular injury in these tubules (Silver Fig 2-Control rat kidney. No significant silver deposit was observed stain, x 150). Fig 3-Kidney from rat given 500 ppm halothane. Exfoliation (Silver stain, x 150). of epithelial cells (arrows) and cellular casts within the lumen of the proximal convoluted tubules (PCT); the collecting duct (CD) appeared to be normal (PAS, X 650). some
9A
'.t 5
e
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Ai%
. j
Fig 4Proximal convoluted tubule (POT) from normal rat. MV=microvilli, BM= basement membrane (X 12,000). Fig 5PCT from rat treated with 10 ppm halothane. Note the membranous swirling of the basal infoldings (arrows) at the base of the epithelial cell. Fig 6PCT from rat given 10 ppm N-nucleus, BM= basement membrane (X 12,000). halothane. Degradation of mitochondria to form large membranous bodies (MB). Remnant of a mitochondria (arrow) could still be recognized (X 17,000).
F
10~ ~
W
~~~~
4--
-%~~~~~~~~~~k Fig 7-PCT from rat treated with 500 ppm halothane. Severe swirling of the basal infoldings. BM=basement membrane, M=mitochondria (x 17,000). Fig 8-PCT from rat administered 500 ppm halothane. Extensive proliferation of the basal infoldings. Displacement of the mitochondria to the apical portion of the cells is also evident. BM=basement membrane (x 12,000).
4k
f
40
'4'~~~~4
J~~~~~~~~~l~~~l
10
-,-w-~~A-
Fig 9-PCT from rat administered 500 ppm halothane. Accumulation of membranous bodies was observed. Many of these bodies were associated with electron-dense particles and became increasingly electron dense. N=nucleus, BM=basement membrane (x 17,000). Fig 10-PCT from rat given 500 ppm halothane. Coalescence of the membranous bodies formed large areas of membranous plaques (MP) within the cell. MV=microvilli (X 17,000).
-@-~~~~~~~~~~~~.
1-41
Fig 11-PCT from rat given 500 ppm halothane. Large areas of membranous plaque (MJP) were seen within the cell; extrusion of the membranous materials into the tubular lumen (TL) was also evident. Large swollen mitochondria in a neighboring cells were also observed. MV-microvilli, N=nucleus (X 12,000). Fig 12-T from rat administered
500 ppm halothane. Extruded membranous materials were seen within the tubular lumen. Increased numbers of Iysosomes and clusters of smooth endoplasmic reticulum (arrow) were also obsenrved. MUV-microvrilli, N=nucleus (X 12,000).
|§W0rlSmq^at.#-:;e5sS$i.,>tl{-X:as@¢.e|84,R1i_7.Sr!~ ~FiqW,BS
.;
L@. .si-t
ii_ VM.eeflq3
._
*K~~~~~~~~~~~~~~~~~~~~~~~~~~~~Win.,s
Xe4it/Sk.t'
2
W
40
*r1r_,xU )= 4z.t} ¢ s^;W,X
':'O'
t'rwe
i$ w 6i;5|F-2.iP .Ae:i.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ s_'s-,, X ,t
"*:B :e F _.
r
f
.s r,
il. ,..:w.
~
;g
tae
A
lage ireual
shpe
(pesmal degrade proein
des
were
~
;_
ta
blody(B
also obere
4 >; It; | ts
s>wasfsfon x 12000_
withi
t - wf Ji>
the
* M je ii,V
epiheia
cl. _
'"
.s'
2.~~~~~~~~~~~~~~~~~~~~~4 4P.~~~
r
~ ~
~
-=
_
t.w..,t,
Fig 15-PCT from rat administered 500 ppm halothane. Accumulation of cytoplasmic dense bodies within a tubular epithelial cell. Many of these cytoplasmic dense bodies appeared to be vacuolated. A large cluster of smooth endoplasmic reticulum (SER) was also observed. N=nucleus (x 26,000). Fig 16-PCT from rat administered 500 ppm halothane. A pyknotic epithelial cell, which became very electron dense, contained many cytoplasmic dense bodies and large swollen mitochondria (x 12,000).
1 7~~.
_iR,wt.
I'D^
,S .=n
.vo
o
.:.S;;^.
_ 7.A
Fig 17-Extrusion of cellular debris, lysosomes and packages of cytoplasmic masses containing mitochondria and other organelles were found within the tubular lumen of many proximal convoluted tubules. MV=microvilli, N=nucleus (X 7750). Fig 18A large package of cytoplasmic mass containing clusters of smooth endoplasmic reticulum (SER), free polyribosomes, and mitochondria was found within the tubular lumen. The microvilli (MV) of the tubules were being compressed by the cytoplasmic mass. N= nucleus (X 17,000). Fig 19-Large cytoplasmic masses within the tubular lumen. A large cluster of smooth endoplasmic reticulum (SER) was included in a cytoplasmic mass. (X 12,000).
2
e20 ;
