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Hepatic alterations following experimental tetracycline toxicity in rats.

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HeDatic Alterations Followina ExDerimental
Tetracycline Toxicity in Rats
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WILLIAM V. ZUSSMAN 1
Department o f Pathology, The Springfield Hospital,
Springfield, Massachusetts
ABSTRACT
Toxic doses of tetracycline were injected intravenously i n adult
female rats to study the morphologic and cytochemical alterations produced in the
liver. Fifty percent of the animals treated died within 48 hours; those surviving were
sacrificed a t 3, 7, 10 or 14 days later. Histologic sections of the livers from all animals
were studied utilizing a specific fluorescence technique to determine tetracycline uptake
and storage, and with histochemical procedures for alkaline phosphatase and succinic
dehydrogenase activity.
Varying degrees of degeneration were observed in the livers of animals dying
acutely. Basophilic lamellated bodies a t seven days and focal venous thromboses a t
14 days were noted in the surviving animals. Tetracycline fluorescence was visualized
initially within bile canaliculi and random hepatic cells, and later within dilated
interstitial spaces as well as i n the basophilic bodies. By 14 days residual fluorescence
was limited to the parenchyma adjacent to thrombosed central veins. Succinic dehydrogenase activity was diminished within areas of tetracycline accumulation and
minimally increased where the cytologic damage was greatest. Alkaline phosphatase
activity present about the central veins and in areas of tetracycline-induced fluorescence reflected parenchymal distortion.
Tetracycline distribution in experimen- the alterations observed with routine histotal animals has been investigated utilizing logic stains, supplementary histochemical
both fluorescent (Bottinger, '55) and enzyme studies of alkaline phosphatase
radiographic (Andre, '65) techniques. The and succinic dehydrogenase activity were
anatomic localization demonstrated, par- carried out.
ticularly after intravenous or intramuscuMATERIALS AND METHODS
lar injection, is not uniform. Consistently
high concentrations are found in the liver,
Twenty-eight female white rats weighwhere following its excretion with the ing 100-150 gm were injected via the
bile, reabsorption from the intestine results right femoral vein with 250 rng/kg of
in further accumulations.
tetracycline hydrochloride previously disIt has been suggested that the gastro- solved in isotonic saline (50 mg/crn3).
intestinal complications observed clinically Livers were removed for study from those
during the course of tetracycline therapy animals that died during the 24-48 hours
(Lepper et al., '51) as well as the hepatic following injection. Surviving rats were
damage often detected (Faloon et al., '57) sacrificed 3, 7, 10 and 14 days later and
are both related to high concentrations of their livers also removed for study. Eight
this drug in the liver.
control rats injected with saline ( 5 cms/
Experiments are undertaken to evalu- kg) were s a c s c e d at the same intervals
ate the hepatic changes produced in rats and their livers studied in a similar
by the administration of known toxic doses fashion.
(LDSo) of tetracycline (P'an et al., '50).
Livers were examined grossly, weighed,
Livers are examined from the animals that
and
20 mm blocks quick frozen in a cryosuccumbed immediately as well as those
stat
at -20°C or placed in either 10%
that survived the single intravenous injection. A fluorescence technique was neutral buffered formalin or acetone at
utilized to localized sites of tetracycline 4°C.
uptake and storage in the microscopic
1 Present address: 1st United States A r m y Medical
sections examined. To evaluate further Laboratory
#1, Fort Meade, Maryland 20755.
ANAT. REC.,162: 301-312.
301
302
WILLIAM V.
The histologic sections prepared from
the formalin fixed tissue were stained with
hematoxylin and eosin. Sections for fluorescence or succinic dehydrogenase studies
were cut at 5 with a cryostat, mounted
on slides, and stored at -30°C.
Fluorescene. Slides were brought to
room temperature and sections mounted
in phosphate buffered glycerine (pH 7.4).
They were then examined under darkfield
illumination with a Reichert fluorescence
microscope using an ultraviolet light source
of 1200 v. The UG1 filter used for excitation resulted in a wave Iength of approximately 365 mu (Regna and Solomons,
'50). Alternate sections were fixed in neutral buffered formalin and stained with
hematoxylin and eosin.
Succinic dehydrogenase. Frozen sections were brought to room temperature
and incubated at 37°C in a substrate prepared as outlined previously by Novikoff
and Essner ('60) containing sodium succinate, nitro-blue tetrazolium and phenazine methosulphate.
Alkaline
phosphatase. The
tissue
blocks fixed in cold acetone were embedded in paraffin. Sections were cut at 3-5 u
and were rehydrated. Gomori's method as
modified by Kabat and Furth ('41) was
then used.
Photograhs were taken using panatomic
film (ASA 32) for light microscopy and
Tri-X pan (ASA 400) for fluorescence.
OBSERVATIONS
Fourteen of the 28 rats treated with
tetracycline died within 48 hours of the
intravenous injection. These animals were
listless and lethargic prior to death. Gross
examination of the livers from these animals showed generalized pallor with focal
areas of hyperemia. In animals surviving
more than 48 hours, lethargy and loss of
appetite during the initial 24 hours were
noted. Pallor of the livers was noted during
the entire two week period of study, the
focal hyperemia being seen only in the first
three days. No change in gross hepatic
weight was present initially, although in
those animals sacrificed at one week an
increase of 6 to 9% was noted. In animals
sacrificed two weeks following tetracycline
injection, the increase was 2 to 4 % .
ZUSSMAN
Initially, variable amounts of degeneration were seen microscopically. Cells were
increased in size and there was cytoplasmic vacuolization (fig. 1 ) . Other cells were
compressed with granular cytoplasm.
There was no change in nuclear morphology. Examinations for succinic dehydrogenase activity showed a normal cytoplasmic distribution and absence of nuclear
enzyme within intact cells (fig. 2). The
cells showing fatty degeneration were almost devoid of enzymatic activity, occasional formazan granules were visible
about their periphery.
The distribution of tetracycline-fluorescence visualized was the same at three
days (fig. 3) as it was in the animals that
died within 48 hours. Pale yellow homogeneous fluorescence was present within
hepatic cells; bright yellow outlined the
bile canaliculi. Foci of fatty degeneration
were non-fluorescent. By three days vacuoles were demonstrated within the cytopIasm of almost all cells (fig. 4). Nuclei
of collapsed cells were visible in many
areas adjacent to intact cells.
By seven days basophilic lamellated
bodies were evident. They were larger than
most nuclei and were seen at random
withjn dilated interstitial spaces (fig. 5).
A pale eosinophilic central area was present in some. By this time tetracycline accumulation within many cells had increased and was non-homogeneous. The
basophilic bodies were highly fluorescent
(fig. 6 ) and the bile canaliculi were dilated, displaying an almost fibrillar yellow
fluorescence. Alkaline phosphatase studies
demonstrated greater than normal activity
in areas of degeneration. The large
amounts of precipitate in these foci (fig. 7)
indicative of enzymatic activity were both
intra- and extra-cellular. Surrounding intact cells demonstrated variable activity;
the nuclear precipitate was coarse to heavy
while that in the cytoplasm was gray or
completely absent. Succinic dehydrogenase
activity was absent in the cells replaced
by fat (fig. 8).
Thrombosed central veins were the most
striking feature noted in the livers of animals sacrificed two weeks following injection (fig. 9). By this time only slight
tetracycline fluorescence was demonstrated. The stores of the drug still present
HEPATIC
ALTERATIONS
IN
were in the central veins and adjacent
controlobular parenchyma (fig. 10). These
areas did not have succinic dehydrogenase
activity (fig. 11). Increased alkaline phosphatase activity was demonstrated in the
parenchyma surrounding thrombi (fig. 12).
DISCUSSION
Most histologic studies of experimental
liver damage demonstrate a variable number of dead, dying, regenerating and otherwise metabolically altered cells (Chang
et al., '58). The interpretation of such
findings is difficult when only ordinary
histologic methods are used. In the present
study the enzymes alkaline phosphatase
and succinic dehydrogenase were evaluated
histochemically in rats that had previously
received toxic doses of tetracycline. This
drug is known to affect enzyme systems
in the liver adversely (Van Meter et al.,
'52; Zimmerman and Humoller, '53). The
tetracycline-induced fluorescence method
utilized made it possible to correlate sites
of uptake and storage with the patterns
of enzyme activity observed.
Helander and Bottinger ('53) were the
first to utilize a histophannacologic technique to study the distribution of oxytetracycline. The strong yellow-green fluorescence of tetracycline derivatives in ultraviolet light, easily differentiated from the
blue and gray-blue autofluorescence seen
in the present saline-injected controls,
facilitated detailed organ and tissue localization.
Andre ('65) studied the distribution in
experimental animals of tritium-labeled
tetracycline, In addition to accumulation
in the spleen, lymphs nodes and skeleton,
the highest concentration was found in
the liver. Excretion in the bile with rapid
passage into the intestine occurred as early
as five minutes following injection, describing a circuit of intestine-to-blood-to-liverto-bile-to-intestine. Intr acellular hepatic
localization, according both to Bottinger
('55) and Andre ('65), is predominantly
centrolobular, the greatest amount being
present in bile capillaries with collections
in large numbers of Kupffer cells.
Reports of liver damage following large
intravenous doses of tetracycline describe
vacuolization and fragmentation of cells
limited mostly to centrolobular areas (Lep-
TETRACYCLINE TOXICITY
303
per et al., '51). Clear, poorly staining water
soluble material crowding out other cytoplasmic elements distend the cells. This
material does not stain for lipid, carbe
hydrate or fat (Cutting, '52). The present
experimentally induced alterations were
histologically similar although predominantly focal, with many intact lobules
present in some animals.
The influence of tetracycline on the development of fatty infiltration in the liver,
according to the review of Shils ('62), is
noted with either relatively large acutely
administered or moderately large long continued dosage. In the present experiment a single toxic dose ( P a n et al., '50)
was administered intravenously in order
to evaluate initial as well as subsequent
changes.
The separation of drug-induced liver injury to groups according to the classification of Popper et al. ('65) represents a
useful practical procedure. Inflammation
characterizes all groups with the exception
of steroid-induced cholestasis and tetracycline-induced steatosis (fatty degeneration). The intracytoplasmic fat globules
seen in the latter group are not associated
with inflammation or necrosis. The fatty
metamorphosis observed in the present experiment was present during the first week
following tetracycline administration. Degenerative changes were suggested later
by the basophilic lamellated bodies seen at
seven days and the intravascular thrombi
at 14 days. In the absence of frank necrosis, the distributoin of the two enzymes
studied histochemically suggested degeneration of the cells replaced by fat during
the first week and subsequent degeneration of cells surrounding thrombosed centrolobular veins after the first week.
DuBuy and Showacre ('61) observed
both in tissue culture and fresh preparations that tetracycline selectively combines
with the mitochondria of living cells. Isolated mitochondria from such preparations
demonstrated decreased oxidative phosphorylation but no changes in oxygen uptake. The localization of tetracycline within
mitochondria were demonstrated by fluorescence.
The relatively decreased centrolobular
succinic dehydrogenase activity normally
demonstrated in the rat liver may be re-
304
WILLIAM V
lated to lower oxygen tension of blood
entering the central vein; the higher activity in the periportal cells suggests oxidative respiration at a higher level. This
heterogeneous enzyme distribution, according to Novikoff ('59), may be involved in
the selective localization of lesions in the
hepatic lobule. The least succinic dehydrogenase activity in the present experiment
was observed in the centrolobular areas,
whereas alkaline phosphatase activity and
tetracycline fluorescence were greatest in
the same zones. It was here that the fatty
metamorphosis was most marked initially
and the basophilic lamellated bodies and
thrombosed vessels were later observed.
Liver cells studied histochemically demonstrate cytoplasmic alkaline phosphatase
activity (Wachstein and Zak, '50). The
enzyme is zonally distributed within the
lobule; the activity is greatest in the cells
about the central vein, intermediate in the
periportal area, and least in the middle
zone. In acute hepatitis Sherlock and
Walshe ('47) found increased activity
within surviving cells. Maximal cell damage occurred in the center of the lobule
where cells demonstrated large quantities
of enzyme. A similar distribution was evident in the livers from animals in the
present experiment that survived the lone&t follow&g tetracycline distribution.
Chemical thrombophlebitis has been
noted in clinical reports of toxicity following intravenous administration of tetracycline (Faloon et al., '57; Lepper et al.,
'51). Thrombosis of hepatic veins were
noted in the present experimental animals
that survived the toxic dose of tetracycline
administered. Flourescent examination of
the thrombosed areas demonstrated large
stores of tetracycline within the vessel
walls as well as the adjacent parenchyma.
Alkaline phosphatase activity was greatest
in these areas both in the animals that
died following tetracycline administration
as well as those with thrombosed veins.
-
LITERATURE CITED
Andre, T. 1956 Studies on the distribution of
tritium-labeled dihydrostreptomycin and tetracycline in the body. Acta Radiol., Suppl. 142:
1-62.
Z U SSMA N
Bottinger, L. E. 1955 On the distribution of
chlortetracycline in the body. Acta Med. Scand.,
151: 343-348.
Chang, J. P., R. E. Stowell, H. E. Betz and M.
Berenbom 1958 Histochemical studies of necrosis of mouse liver in &TO.
Arch. Path., 65:
479-487.
Cutting, W. C. 1952 Liver changes produced
by aureomycin and terramycin. Sanford Med.
Bull., 10: 19-20.
DuBuy, H. G., and J. L. Showacre 1961 Selective localization of tetracycline in mitochondria
of living cells. Science, 133: 196-197.
Faloon, W. W., J. J. Downs, K. Duggan and J. T.
Prior 1957 Nitrogen and electrolyte metabolism and hepatic function and histology in
patients receiving tetracycline. Am. J. Med.
Sci., 233: 563-572.
Helander, S., and L. E. Bottinger 1953 On the
distribution of terramycin in different tissues.
Acta Med. Scand., 148: 71-75.
Kabat, E. A., and J. Furth 1941 Histochemical
study of distribution of alkaline phosphatase in
various normal and neoplastic tissues. Amer. J.
Path., 17: 303-318.
Lepper, M., C. K. Wolfe, E. R. Caldwell, Jr., H. W.
Spies and H. F. Dowling 1951 Effect of large
doses of aureomycin o n human liver. Arch.
Intern. Med., 88: 271-283.
Novikoff, A. B. 1959 The biochemical cytology
of the liver. Bull. N. Y. Acad. Med., 35: 67-70.
Novikoff, A. B., and E. Essner 1960 The liver
cell. Some new approaches to its study. Am. J.
Med., 29: 102-131.
Pan, S. Y., L. Scaduto and M. Cullen 1950
Pharmacology of terramycin in experimental
animals. Ann. N.Y. Acad. Sci., 53: 238-244.
Popper, H., E. Rubin, D. Gardiol, F. Schaffner
and F. Parnotto 1965 Drug-induced liver
disease. A penalty for progress. Arch. Intern.
Med., 115: 128-136.
Regna, P. P., and I. A. Solomons 1950 The
Chemical and physical properties of terramycin.
Ann. N. Y. Acad. Sci., 53: 229-237.
Sherlock, S., and V. Walshe 1947 Hepatic alkaline phosphatase: histological and microchemical studies on liver tissue i n normal subjects
and in liver and bone diseases. J. Path. Bact.,
59: 615-630.
Shils, M. E. 1962 Some metabolic aspects of
tetracyclines. Clin. Pharm. Therapeut., 3: 321339.
Van Meter, J. C., A. Spector, J. J. Oleson and
J. H. Williams 1952 In vitro action of aureomycin o n oxidative phosphorylation in animal
tissues. Proc. SOC.Exp. Biol. Med., 81: 21521 7.
Wiihstein, M., and F. G. Zak 1950 Alkaline
phosphatase in experimental biliary cirrhosis.
Am. J. Clin.Path., 20: 99-115.
Zimmerman, H. J., and F. L. Humoller 1953
Effect of aureomycin on choline oxidase and
other enzyme systems of rat liver. Am. 1.
Physiol., 175: 468-472.
~~
PLATES
PLATE 1
EXPLANATION OF FIGURES
306
1
Degeneration observed i n liver of rat that died 24 hours following intravenous tetracycline administration. Altered cells show foamy vacuolated cytoplasm; intact cells are compressed. Hematoxylin and
eosin, x 290.
2
Intact liver cells from the same animal as figure 1 show a normal
cytoplasmic distribution of succinic dehydrogenase activity. Areas
free of activity correspond to damaged cells. Few coarser granules
are present in adjacent foci. Nitro-blue tetrazolium, x 290.
3
Tetracycline localization in liver of rat sacrificed three days following
intravenous injection. Coarse and fine fluorescent fibers outline the
bile canaliculi. Hepatic cells are faintly fluorescent. Darkfield illumination with a n ultraviolet light source of 365 mF, x 290.
4
Degeneration observed in liver from same animal as figure 3. Cytoplasmic droplets are present i n most cells. Hematoxylin and eosin,
X 290.
HEPATIC ALTERATIONS IN TETRACYCLINE TOXICITY
William V. Zussman
PLATE 1
307
PLATE 2
EXPLANATION OF FIGURES
5 Dark-staining lamellated bodies present in liver examined from animal sacrificed seven days following intravenous tetracycline administration. Intact liver cords are compressed. Hematoxylin and eosin,
X 290.
308
6
Tetracycline distribution in liver from same animal as figure 5. The
lamellated round bodies are highly fluorescent. Fine and coarse fibers
outline the bile canaliculi. Darkfield illumination with a light source
of 3 6 5 m p , x 380.
7
Alkaline phosphatase activity in liver from same animal as figure 5.
Most of the precipitate, both intra- and extracellular, is observed (upper left) in areas of fatty replacement. Adjacent cells show variable
nuclear and cytoplasmic activity. Gomori’s method as modified by
Kabat and Furth (’41),X 290.
8
Area devoid of succinic dehydrogenase activity (left center) corresponds to focus of increased alkaline phosphatase activity seen in
figure 7. Adjacent intact cells show variable amounts of precipitate.
Nitro-blue tetrazolium, X 290.
HEPATIC ALTERATIONS IN TETRACYCLINE TOXICITY
William V. Zussman
PLATE 2
PLATE 3
EXPLANATION OF FIGURES
9 Thrombus in central vein of liver from rat sacrificed 14 days following intravenous tetracycline administration. Venous wall is incorporated into the partially organized thrombus. Hematoxylin and
eosin, x 240.
10
Tetracycline distribution i n area surrounding a thrombosed vein in
same animal as figure. 9. Coarse fluorescent fibers outline the venous
wall. The thrombus is lightly fluorescent. Fine fluorescent fibers outline the bile canaliculi. Darkfield illumination with a n ultraviolet
light source of 365 mp, X 290.
11 Distribution of succinic dehydrogenase activity in liver surrounding
thrombosed central vein. Liver from same animal as figure 9. This
area (lower) is devoid of activity. Some of surrounding cells show
increased acitvity. Nitro-blue tetrazolium, x 240.
12 Alkaline phosphatase activity in a similar area as figure 11 from same
animal as figure 9. Increased intra- and extracellular precipitate is
present adjacent to the thrombosed vein (lower left). Gomori’s method
as modified by Kabat and Furth (’41), x 240.
310
HEPATIC ALTERATIONS IN TETRACYCLINE TOXICITY
William V. Zussman
PLATE 3
311
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