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Morphologic effects of minocycline in laboratory animals
TOXICOLOGY
AND
APPLIED
Morphologic
PHAR.vIACOLOGY
11,
150-170
Effects of Minocycline
( 1967 )
in laboratory
Animals
K.-F. BENITZ, G. K. S. ROBERTS,AND A. YUSA Toxicology
Research Section, Lederle Laboratories Division, American Pearl River, New York 10965 Received April
Cyanamid
Compa1~~4,
10, 1967
The synthesis of minocycline, a new antibiotic of the tetracycline group, was reported by Martell and Boothe ( 1967). The compound is chemically defined as 7-dimethylamino-6-demethyl-6-deoxytetracycline. Redin ( 1967) has shown that this compound has unique and potentially useful antibacterial properties. The results of studies on drug metabolism and tissue distribution of this compound have been reported by Kelly and Kanegis ( 1967). Short-term toxicity experiments and observations of certain aspects of the pharmacology of minocycline have been reported by Noble et al. ( 1967). Our studies report the nature and the degree of morphologic effects of this drug in several species of laboratory animals after short-term administration of the drug. Long-term toxicity studies are in progress and the results will be reported at a later date. MATERIAL
AND
METHODS
Rats ( Sherman strain), dogs (purebred beagles), monkeys (Cercopitlrecus aetIriops), and mice (CF, albino) were used. At the end of each experiment the nonrodents were sacrificed by intravenous injection of sodium pentobarbital whereas chloroform inhalation was used for rodents. In two studies (One-Month Study in Rats and One-Month Study in Dogs) complete autopsies with inspection of all three body cavities and determinations of body weights and organ weights were performed. Paired organs were weighed together, and the weight of the thyroid gland was obtained after fixation. Representative samples of all major organs (heart, trachea. lungs, tongue, salivary glands, esophagus, stomach, small and large intestines, liver, gall bladder, pancreas, kidneys, urinary bladder, thymus, spleen, mesenteric lymph nodes, femoral marrow, pituitary gland, thyroid gland, adrenal gland, gonads, seminal vesicles and prostate, uterus, femur, skin, eyes, brain, and spinal column) were placed in suitable fixatives (4% aqueous formaldehyde, 8% neutral formaldehyde, and Bouin’s, Zenker’s? SUSA, and Helly’s solutions ), and paraffin sections from this material were used for a variety of conventional staining methods (hematoxylin and eosin, luxol-fast-blue-cresyl-fast-violet, Masson’s trichrome method, and iron reaction). In some instances unstained paraffin and cryostat sections were examined with a phase-contrast system. The following histochemical procedures were used for thyroid gland material: Turnbull’s reaction (Mallory, 1942), periodic acid-Schiff stain (Lillie, 1948), Nile blue stain ( Lillie, 1956), Nile bl ue stain (Hueck, 1912), Schmorl’s method for lipofuscins 150
MORPHOLOGIC
EFFECTS
OF
MINOCYCLINE
151
(Pearse, 1960), Ziehl-Neelsen stain (Pearse, 1960), ferrous iron reaction ( Barka and Anderson, 1963), methenamine silver method (Barka and Anderson, 1963), and bleaching with HaOz or peracetic acid (Pearse, 1960). Thyroid gland material of animals selected from the high dose groups of these rat and dog studies was examined by fluoresence microscopy. Similar techniques were used for additional studies; however, the morphologic examinations were limited to the thyroid glands. Samples for electron microscopy were removed quickly from the animal under light chloroform anesthesia and were fixed in cold (O-5” ) 1% osmium tetroxide in 0.1 M phosphate buffer pH 7.35 for 60 minutes, dehydrated, cleared in styrene, and embedded in Vestopal W. Additional samples were also fixed in cold (O-5” ) 5.0% glutaraldehyde in 0.1 M phosphate buffer pH 7.35 for 2 hours followed by 20 minutes in cold 1% osmium tetroxide, dehydrated in alcohol, cleared in propylene oxide and embedded in Maraglas-Cardolite. Some samples fixed in glutaraldehyde were embedded in Maraglas-Cardolite without postfixation in osmium tetroxide for comparison with other preparative procedures. Silver and light gold sections stained with lead citrate or uranyl acetate were examined using magnifications ranging from 3000 X to 30,000 X. Numerical data from all studies were examined for significant differences among groups using the rank test method of Wilcoxon ( 1945, 1947). Some morphologic changes were graded using as scores: absent, doubtful, slight, moderate, and marked. RESULTS
One-Month
Study in Rats
Eighty animals of both sexes were used for this study. Twenty rats served as controls whereas 3 other groups received 75, 25, and 8 mg/kg/day of minocycline by gavage for 31-35 days. Complete histologic examinations were carried out on the high dose group and the control animals. Observations during life have been reported by Noble et al. ( 1967). No morphologic changes were found that could be attributed to treatment except in the thyroid glands and the skeleton. The findings in the thyroid glands are summarized in Table 1 and illustrated in Fig. 1. It is obvious from these data that the oral administration of minocycline caused a marked black discoloration sometimes associated with hyperplastic changes (morphologic activation). This discoloration was dose related and was present in all treatment groups. A dark pigment was also seen histologically. Although some insignificant pigment granules were normally present in the glands of controls, the drug-induced pigmentation was much more pronounced, It was more conspicuous in follicles with high epithelium than in others that were lined with low cuboidal cells. It consisted of nonfluorescent dustlike particles (average diameter approx. l-2 p) of brown-black color (depending on the thickness of the sections) and was predominantly located in methods that the apical portion of the follicular epithelium. The histochemical were applied to some thyroid specimens gave inconclusive results. The Turnbull
(g):
(mg):
a Values in parentheses c Significantly different
IO/l0 -
IO/l0 -
lO/lO lO/lO -
lo/lo -
lo/lo -
TABLE
1
different
from
control
5/10 5/10
lo/lo
Y/10 7/10
7/10 3/10
l/l0 Q/l0
l/l0 Q/l0 -
Q/l0 l/l0
F 206 (186-232) 19.6 (17-23) 9.5 (7.8-11.0)
e/10 s/10
lO/lO -
6110 J/10
M 303 (23&349) 25.8C (20-35) 8.5c (6.6-10.8)
75 mg/kg/day
OBTAINED FROM MALE AND FEMALE (BABE) ORALLY FOR ONE MONTHQ
b Significantly I 6 0.5).
-
IO/l0 -
lo/lo -
-
lO/lO
F 216 (189-246) 19.1 (17-23) 8.9 (6.9-10.6)
GLANDS
lo/lo -
group(P
0 (Controls)
IN THYROID
M 284 (249-314) 20.9 (17-27) 7.3 (6.6-8.6)
FINDIN(;S
= ranges. from control
Relative weights (mg/ 100 g body weight):
weights
weight
Absolute
Sex: Body
Dose:
OF MORPHOLOGIC
Gross findings a. Size Normal Slightly enlarged b. Color Pink Black Dark brown Brown Red brown Dark red c. Isthmus Invisible Prominent Microscopic findings a. Hyperplastic changes Absent Slight b. Pigmentation Absent Slight Moderate Marked
SUMMARY
25 w/Wday
RECEIVING
group
(P
IO/10
5 0.01).
3 / 10 7/10
S/l0 7/1n
5110 5/10
Q/l0 l/10
M 3%‘b (W-385) 23.6 (a&29) 7.3 (5.2-8.7)
RATS
a/10 s/10 -
Q/l0 l/l0
3/10 7/10
4/10 l/IO 5/10 -
Q/10 l/10
F 207 (19&223) 18.4 (16-23) 8.9 (7.9-10.3)
VARI~C~
DOSE
OF MINOCYCLINE
R/l0 ?/IO !I/10 l/l0 -
8/10 e/10
7110 3/10
6jlO 4110
9110 l/10
2/10 S/l0
lo/lo
F 207 (181-231) 16.8 (12-22) 8.1 (7.3-10.0)
8 mdkgldw
3/10 2jlO 5/10
lo/lo
M 323b (264-371) 24.4 (19-31) 7.6 (6.1-9.1)
LEVELS
MORPHOLOGIC
FIG. 1. Morphologic male, 75 mg/kg/day fication: X 680.
EFFECTS
OF
153
MINOCYCLINE
activation with marked pigmentation of minocycline per OS for 35 days.
of thyroid gland. Rat Formalin, hematoxylin-eosin.
No.
K 147, Slagni-
reaction was negative whereas both Nile blue stains and the Schmorl procedure gave positive results as with lipofuscins. The Ziehl-Neelsen stain for acidfast lipofuscins was negative. Both methods for the detection of melanins, ferrous iron method and methenamine silver technique, were negative whereas exposure to Hz02 (48 hours) or peracetic acid (16 hours) resulted in bleaching. The PAS stain showed a reddish-brown reaction site, presumably a combination of black pigment with a red-stained polysaccharide component indicating that this material was of colloid nature. The periodic acid Schiff reaction gave a distinct brown color that was interpreted as a combination of black pigment and magenta colloid material of polysaccharide nature. The morphologic activation consisted of proliferation of the follicular epithelium associated with an increase in cell height, decrease in stainable colloid, and capillary hyperemia. These slight hyperplastic changes that were present in all treatment groups, as shown in Table 1, were not associated with any changes in serum protein-bound iodine values (Noble, et al., 1967). One could, therefore, conclude that the oral administration of minocycline did not cause any marked hypothyroidism in this species. A summary of the morphologic observations covering the changes in the skeletal system is given in Table 2. The measurements of the femur length indicate that the administration of minocycline did not result in any impairment of
54
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S. NOBERTS,
AND
A.
YUSA
MORPHOLOGIC
EFFECTS
OF
MINOCYCLINE
155
skeletal growth. This was confirmed by the microscopic examination of the distal epiphysis using various criteria such as the width of the epiphyseal disk, the arrangement of cartilage cells, the amount of trabecular bone, and the thickness of the cortex. Some of the femora from the high dose group were examined grossly under ultraviolet light (3660 A) and although they were distinctly yellow at autopsy, no fluorescence was seen. All other pathologic findings consisted of disease entities known to occur in rats, and no effects of treatment on incidence, nature, or degree of these spontaneous diseases were noticeable. One-Month
Study in Dogs
Ten beagle hounds were used and divided into 5 groups (1 male and 1 female per group). Doses of 40, 20, 10, and 5 mg/kg/day of minocycline were given by the intravenous route for 27-29 days. The control animals received saline. The results of observations during life have been reported by Noble et al. (1967). The intravenous administration of minocycline caused the following toxic effects: (1) morphologic activation and pigmentation of the thyroid gland together with formation of thyroidectomy cells in the pituitary gland and (2) siderotic changes consistent with and attributable to intravascular hemolysis. In the two animals receiving the highest dose (40 mg/kg/day) these changes were accompanied by the formation of occasional bile plugs. The findings in the thyroid gland together with the changes in the pituitary gland are given in Table 3. The nonfluorescent pigment was grossly and microscopically not clearly dose dependent. As shown in Fig. 2 in some instances larger clumps of presumably the same pigment were present in the colloid. The morphologic activation was present in 3 of 4 animals of the two high-dose groups. This was mediated by the pituitary gland as evidenced by the presence of thyroidectomy cells. The amount of hemosiderin in spleens, livers, and kidneys of the animals receiving 40 or 20 mg/kg/day of minocycline was presumably attributable to intravascular hemolysis (see Table 4). According to Noble et ~2. ( 1967) a dose of 10 mg/kg/day caused only very slight hematologic changes that were almost within the range of normal fluctuation. However, in view of the hematologic findings in the animals of the 40 and 20 mg/kg/day groups, these changes could be considered as suggestive of a slight degree (trace) of hemolytic anemia although they were not accompanied by any abnormal siderotic changes except for a moderate increase in iron-positive pigment in the spleen. The presence of occasional bile plugs does not necessarily indicate hepatic (elevated sulfobromophthalein retencell damage since, with one exception tion), all liver function tests were within normal ranges (Noble et al., 1967). In addition, no morphologic changes indicative of hepatocellular damage were evident. The drug-induced erythema as described by Noble et al. (1967) was not found at autopsy or in microscopic preparations. This is not surprising since
body
FINDIKGS
also in colloid.
M F M F M F M F M F
Sex
OF MOHPHOLOQIC
0 Pigmentation
5
10
PO
40
0
Dose (Wkiday)
SVMMARY
7.3
9.7
7.2
8.2
6.3
1.3 0.7
0.9 1.2 0 6 0.8 0.9
6.5
7.2 9.5
(6’) 0.5 0.6 0.6
-
weight
.4bsolute
GLAXDS
8.1 9.4
Final weight (kg) -
IX THYROID
TABLE
3
findings
96
134
125
95 98
125 126
9%
62 64
Relative weight (w/k)
Gross
Pale pink Pale pink Black Black Black Black Black Black Gray Black
Color at autopsy Absent Absent Marked Absent Moderate Marked Moderate Moderate Slight Slight
Hyperplasia
Microscopic
OBTAINED FHOM MALE AND FEMALE Dots RECEIVING (Rasp) INTHAVENOUSLY FOR ONE MONTH
DOSE
Absent Absent Marked” Slight Moderate Moderate” Slight Slight Slight Moderatea
Pigmentation
findings
V4~ror-s
Absent Absent Present ilbsent Present Present hbsent Absent dbsent .4bsent
Thyroidectomy cells in pituitary gland
T,EVCLS OF MIXWYPLINE
MORPHOLOGIC
erythema belongs to the transient appear immediately after death. One-Month
Study
EFFECTS
capillary
OF
157
MINOCYCLINE
phenomena
that are known
to dis-
in Monkeys
It was, of course, of interest to determine whether these thyroid gland changes could also be found in other species. Since both the rat and the dog reacted with morphologic activation and pigmentation, an additional experiment in African green monkeys was carried out to determine whether pig-
FIG. apical colloid.
2. Marked morphologic portion of follicular Dog P 4494, male,
activation of thyroid gland epithelium and heavy pigment 40 mg/kg/day of minocycline
with fine granular pigmentation in deposits within barely stainable intravenously for 30 days.
mentation occurred and was associated with hyperplastic changes. Observations during life have been reported by Noble et al. ( 1967). Three male and 3 female monkeys received 30 mg/kg/day of minocycline per OS, in gelatin capsules for 30 days. Two female animals served as controls. All animals survived the experimental period with the exception of 1 male that died spontaneously after receiving 22 doses. The cause of death was not determined. The morphologic examination was limited to the thyroid glands. The results are given in Table 5. These data indicate that monkeys reacted slightly differently from rats and dogs. Although all thyroid glands of the treated group were grossly discolored, only one half of the animals showed pigmentation microscopically. There was no morphologic activation associated with these
158
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BENITZ,
G. K.
S. ROBERTS,
AND
A.
YUSA
changes. In general, the degree of pigmentation was much less than in the dog, the other nonrodent species that could be used for comparison. One could speculate that the lack of an active hyperplastic process could have been responsible for the inconsistent reactivity pattern with respect to pigment deposition.
of
Mode
Action
Study
in Rats
These experiments showed that minocycline caused hyperplastic changes and dark pigmentation in the thyroid glands of various species. Many antithyroid drugs, for instance the thioureas, are believed to act by inhibiting a peroxidase system necessary for the oxidation of inorganic iodine prior to iodination of tyrosine. This mechanism of action and the observations made by Kelly and Kanegis (1967) that minocycline is easily oxidized to an insoluble black substance suggested that it might be a competitive inhibitor of iodine peroxidase, being oxidized to a black pigment in the process. If this were the case, one might expect an antithyroid compound such as propylthiouracil to inhibit the peroxidase system and therefore to prevent the oxidation of minocycline and thus the T.4BLE SUMMARY BILE
OF DEGREES
PLUGS
OF ANEMIA,
OBSERVED
IN
ORGAN
4
SIDEROSES,
DOGS RECEIVIKG TNTRAVENOT-SLY
AND
PRESENCE
Siderosis Dose (mg/kg/day)
Sex
0
M F M F M F
40 20
F
Absent Absent Moderate Moderate Slight Slight Trace Trace
details,
see Noble
M
10
(1 For
Degree of anemia”
hematologic
Slight Slight Marked Marked Moderate Moderate Moderate Moderate
SUMMARY
OF MORPHOLOGIC! RECEIVING
FIXDINGS
30 MG/KG/DAY
of Kidney
Slight Slight AMarked Marked Slight Moderate Slight Slight
Absent Slight Marked Moderate Absent Slight Absent Slight
Controls Treated
Gross
GL~XDS
OBTAINED
ORALLY OF MINOCYCLINE ITPITH CONTROLS
IN THYROID
(BASE)
findings
Normal pink with invisible isthmus Dark red-brown Brown Black 5/6 Prominent isthmus Invisible isthmus
Hepatic hile plugs Absent Absent Present Present Absent .4bsent ;\hsent
5 FROM
Hyperplasia
6 MALE
FOR 30 Ds~s
Microscopic Group
(BOSE)
(1967).
TABLE MONKEYS
OF HFPATI~
OF MINOCYCLINE
Liver
Spleen
et al.
OR ABSENCE
V.~RIOUS DOSE LEVELS FOR ONE MONTH
.\s\‘u
FEMALE
IN COMPARISON
findings Pigmentation
Absent
?ja
Absent
2b
Absent
616
Doubtful
3/6
Slight Moderate
‘3/6 l/6
%ja
I/‘3 5/6 l/6
MORPHOLOGIC
EFFECTS
OF
MINOCYCLINE
159
pigment deposition. The same inhibitory process should still cause a morphologic activation of the gland. Conversely, if the enzymatic activity of the gland could be suppressed by an exogenous thyroid substance, one could expect the prevention of the minocycline-induced morphologic activation and also the pigmentation of the gland. This hypothesis was tested in the following experiment. Eighteen male Sherman rats (4 or 5 per group) were treated for 1 month. One group served as controls. A second group received minocycline at a standard dose of 100 mg/ kg/day for 5 days per week by gavage. The third group received the standard dose of minocycline and a 1.0% drug diet of thyroid powder USP (average intake approximately 800 mg/kg/day ), Propylthiouracil USP was given to a fourth group in drinking water as an 0.01% solution (average intake approximately 8 mg/kg/day) along with the standard dose of minocycline. The results of this experiment are given in Table 6. Again, minocycline alone caused a deposition of pigment and moderate morphologic activation of the thyroid glands in all animals. However, the simultaneous administration of minocycline plus propylthiouracil resulted in a marked degree of hyperplastic changes without any pigmentation. These observations suggest that thyroid glands undergoing rapid hyperplastic and hypertrophic changes were not able to form any pigment presumably derived from minocycline. The administration of minocycline plus thyroid powder resulted in an extreme storage phase and again no pigmentation was found. Since the administration of thyroid powder obviated the need for an active synthesis of thyroid hormones, this hypotrophic state of activity also interfered with the formation of minocycline-induced pigmentation. These results confirm the statement that minocycline has an antithyroid effect and support the speculation that enzyme activity in the gland is necessary for the formation of the pigment. The most likely system seems to be an iodide peroxidase, and minocycline itself is the likely source of the pigment. Onset and Recovery
Study
in Rats and Mice
Preliminary experiments indicated that the pigmentation of thyroid glands occurs rather early, especially in rats. It could be tentatively identified 2 weeks after oral administration of 100-200 mg/kg/day. It was therefore of interest to study the early phases of pigmentation using electron microscopy. Fifteen adult rats were divided into 3 groups (5 animals per group). Three rats received 100 mg/kg/day of minocycline intraperitoneally; 2 animals served as controls. These groups were sacrificed at weekly intervals (after 1, 2, and 4 weeks of drug administration). In addition 2 animals received 144 mg/kg/day of minocycline orally and were sacrificed after 26 days of treatment. After 1 week of treatment, no ultrastructural differences between control and experimental animals were found. After 2 weeks of treatment a considerable number of black bodies was found in approximately 50% of the follicular cells. This change became more pronounced after 26-28 days of treatment and was usually confined to the apical cell region (Figs. 3 and 4). These structures were usually spherical or oval (approximately 0.1-1.5 p in diameter) and
0 Values in parentheses b In drug diet. c In drinking water.
4
Minocycline + 1.0% thyroid powderb Nnocycline + 0.01 y0 propylthiouracilC
= ranges.
5
4
Number of rats
ALONE
Knocycline
MONTH
&VDISGS
5
Treatment
ONE
OF MORPHOLOGIC
Controls
SUMMARY
THYROID
282 (26+316) 287 (266-306) 271 (26CH86) 238 (214-264)
k)
Body weight
OR IN COSJZ~NCTION
IS WITH
GLANDS
(77YO2)
(16::P)
(25Z3)
(266)
(md
Absolute weight
THYROID
OBTAINED POWDER
FROM
T$BLE
6 RECEIVIKG
findings
8.3 (7.6-9.7) 9.9 (8.2-10.9) 7.4 (7.0-7.7) 38.1 (29.2-44.4)
Relative weight (mg/lOO g B.W.)
Gross
100
Red
Pink
Black
Pink
515
4/J
414
515
Microscopic
5/5 Moderate hyperplasia 4/P Extreme storage phase 414 Marked hyperplasia 515
Normal
(BASE) CONTROLS
AMorphologic state
WITH
OF MISOCYCLIKE
IN COMPARISON
MG/KG/D.IY
Color at autopsy
_____
OR PROPYLTHIOUR.~CIL
RATS
.4hsent
Bbsent
Present
absent
Pigmentation
findings
ORALLY
FOH
MORPHOLOGIC
EFFECTS
OF
MINOCYCLINE
161
FIG. 3. Numerous electron dense bodies (DB) of varying dimensions, some of which show aggregates of darkly stained particles. A large, rather homogeneous colloid droplet (CD) of lighter density is present. M = rather prominent mitochondria; ER = ergastoplasm; h4V I microvilli; L = lumen. Thyroid gland of rat No. B 3906, female, 144 mg/kg/day of minocycline orally for 26 days. Five percent glutaraldehyde followed by 1.0% osmium-tetroxide. Magnification: X 19,950.
showed a homogeneous dark grayish black opacity after glutaraldehyde fixation (see Fig. 5). Postfixation with osmium followed by lead staining or lead staining alone after glutaraldehyde fixation resulted in the appearance of intense grayish black to dark black bodies as shown in Fig. 6. They consisted of a homogeneous matrix sometimes containing fine fibrous or threadlike elements,
162
K.-F.
BENITZ,
G.
K.
S. ROBERTS,
AND
A.
YUSA
FIG. 4. Thyroid epithelium with nmI,erous small colloid droplets (CD) of medium density are dispersed among the ergastoplosm ( EH) containing dilated cisternae. N = nucleus: AIV = microvilli; L = lumen. Thyroid gland of rat No. B 3910, female, control. Five percent glutaraldehyde followed 1))~ 1.0% osmium-tetroxide. Magnification: x 19,950.
ringlike structures about 0.05 p in diameter with a light center and a dark rim, and some dense particles of various dimensions. These bodies appeared to have a membrane, and in some sections a thin tenuous line delineated these structure from the surrounding cytoplasmic matrix. The mitochondria were somewhat more pronounced than normal. Nuclei, endoplasmic reticulum, and Golgi entities were found complex did not show any alterations. No novel structural in the follicular epithehum. The morphologic characteristics of the dark bodies with respect to size, shape,
MORPHOLOGIC
FIG. without bodies female,
EFFECTS
OF
MINOCYCLINE
163
S. Electron dense bodies obtained from a thyroid specimen fixed in glutaraldehyde postosmification or heavy metal staining. Please note the natural opacity of these as well as the network of dense material within these aggregates. Rat No, B 390’7, 144 mg/kg/day of minocyclinc orally for 26 days. Magnification: x 51,3011.
Frr.. 6. Dense bodies containing typical ringlike elements. Thin membranes (MB) were found in close apposition to these bodies. Thyroid gland of rat No. B 3906, female, 144 mg/kg/day of minocycline orally for 26 days. Five percent glutnraldehyde followed by 1.0% osmilun tctroxide and stained with uranyl acetate and lead citrate. Magnification: x ,51,500.
164
K.-F.
BENITZ,
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K.
S. HOBERTS,
AND
A.
YUSA
and distribution did not differ from the structures believed to be precursors of colloid droplets themselves except for their increased opacity and stainability. All other alterations were consistent with changes found in stimulated thyroid glands as described by Ekholm ( 1964). A similar time-sequence study was carried out in mice. Twenty animals were used for this study and 250 mg/kg of minocycline was administered orally for 28 days. Two treated and 2 control animals were sacrificed on days 3, 7, 14, 21, and 28 of treatment. The morphologic examination was limited to gross inspection which proved to be sufficient for the detection of pigmentation in other species. The neck regions of the animals were placed under a dissecting scope and the thyroid glands were exposed and inspected in situ for changes in size and color. Then the glands were removed together with the trachea and larynx and reexamined at a higher magnification. No difference in color or size between control and experimental animals was found although a rather high dose was used. These findings were in contrast with the observations made in rats, dogs, and monkeys, Except for possible species differences, this discrepancy cannot be explained at the present time although good absorption by mice was obtained in similar experiments ( Redin, 1967). The possible regression of pigmentation and hyperplastic changes was studied in 27 rats that received 75 mg/kg of minocycline for 38 days and were then kept for 1 year on a normal diet. An equal number of animals were kept as controls. The morphologic examination was limited to the thyroid gland. The results of this study are given in Table 7. Gross and microscopic findings indicated that the pigmentation was still present after 1 year of recovery. The slightly increased pigmentation in the control group in comparison with similar findings from the 1 month experiment was not surprising. These rats were about 14 months old, and it is known that the thyroid gland shows increased amount of pigmentation with age. Differences in microscopic appearance also helped to distinguish between treated and controls. The pathologic pigment usually consisted of coarse granules whereas the follicular epithelium of the control animals contained considerably fewer granules of smaller particle size. In all treated animals some pigment deposits were also found in the colloid, usually located next to the epithelial border. Colloid storage and follicular pattern were of the same degree and incidence in both treated and control groups. No signs of morphologic activation were found. One can therefore conclude that the pigment itself is probably not the cause of the hyperplasia of the thyroid gland as observed at the end of the l-month experiment. The presence of this unknown pigment did not change the gross and histologic characteristics of the glands. One can assume that once this pigment is deposited within epithelium and colloid, it remains there for a long time. However, the morphologic findings point to the fact that the presence of this foreign substance is apparently innocuous. DISCUSSION
The hemolytic anemia observed in dogs was rather mild and did not result in severe hemolytic crises, nor was it associated with jaundice. According to
MORPHOLOGIC
EFFECTS
TABLE ~I!MMARY RE~EIVED'~~
OF
&~ORPHOLOGIC MG/KG/DAYOF
FIKDINGS IN THYROID MIKOCYCLINE(B.ME)ORALLY
PERIOD
IN COMPARISON
OF
165
MINOCYCLINE
7 GL.4NDS OBT.4INED FHOM R.j'rs HAVING FOLLOWED BY A ONE-YE.W RECOVERY WITH
CONTROLP
Treated
Controls
Absolute
weights
(ma):
Cross findings a. Size Normal Questionably enlarged 1. Color Pink Black Brown-black Dark brown Dark pink Microscopic findings a. Colloid storage Absent Slight Moderate Marked b. Follicular pattern* Macro Mixed Micro c. Pigmentation’ Absent Slight Moderate Marked
Males 29.3 (25-35)
Females 22.1 (19-25)
lY/lS -
14114
11/13 -
14/14
AMales si.5 (41-36)
Females 25.0 (14-35)
13/13 -
13/14 l/IS -
lo/l3 Y/13 -
Y/13
7114 3/14 4114
2114
-
-
4113 6/13 3/13
6114 6114
4113 6/13
“2114
Y/l3
l/14 S/l4 7114 l/14
S/I3 7113 l/l3
8114 S/l4 -
o/13 Y/l3 2113
7114 6114 l/14
8114 5114 l/l4
-
u Values in parentheses = ranges. b Macro = predominantly macrofollicular terns interspersed; micro = predominantly c Based on phase contrast microscopy.
S/l3 l/l3 l/l3
pattern; mixed microfollicular
l/l4 4114 9/14
4/13 9113
= macro.pattern.
and
microfollicular
pat-
Noble et al. (1967) there was no visible evidence of drug-induced hemoglobinemia or hemoglobinuria. Usually, a hemolytic process of a subacute nature does not impair hepatic function (Popper and Schaffner, 1957). However, the increased sulfobromophthalein sodium retention observed in the two animals receiving 40 mg/kg/d ay was considered by Noble et al. (1967) as indicative of early hepatocellular malfunction. However, no morphologic evidence was found to substantiate this interpretation. The intrahepatic cholestasis may account for this functional change since according to Hallesy and Benitz (1963) hepatic siderosis per se does not cause abnormal BSP retention values. A dose of 5 mg/kg/day did not cause any hemolytic anemia nor did it cause any abnormal deposition of iron-positive pigment in spleen, liver, and kidneys.
166
K.-F.
BENITZ,
G. K.
S. ROBERTS,
AND
A.
YUSA
Therefore, these animals were excluded from Table 4. According to Wintrobe ( 1961) an anemia of similar type that was not accompanied by active regeneration of red cells has been found in cases of spontaneous hyperthyroid&m and also after the total ablation of the thyroid gland in man and laboratory animals. The yellow discoloration observed in the femur and calvarium of all treated rats was not surprising because it is well known that tetracycline derivatives are rapidly deposited in bony structures. Some of the bones were examined under ultraviolet light but did not show a yellow fluorescence in spite of the fact that they appeared yellow in ordinary light. Apparently the calcium chelate complex of minocycline did not fluoresce but Kelly and Kanegis (1967) have shown that magnesium forms a fluorophore with this compound. However, the femur length and microscopic examination of the femora from the 75 mg/kg/ day group did not show any difference from controls. These findings suggest that minocycline does not cause an impairment of postnatal skeletal growth, a phenomenon claimed by Cohlan et al. ( 1961, 1963) for tetracycline but unconfirmed by observations made in our laboratories. If any harmful effects had taken place one would have observed an effect in these rodents since the experiment was started 1 week after weanling age and continued during a period of rapid skeletal growth. The most striking drug effect was the abnormal pigmentation of the thyroid gland that was observed in rats, dogs, and monkeys. The rat and the dog reacted also with a hyperplastic process which was not detectable in monkeys. The hypothyroid condition of dogs treated with minocycline could already be suspected in viva since according to Noble et al. (1967) the serum proteinbound iodine (PBI) values decreased to 3042% of the predose values. However, in one of the high dose animals the morphologic activation was absent and no thyroidectomy cells were found in the pituitary gland. This inconsistency was probably not surprising since the usefulness of the PBI determinations in dogs is stil1 to be determined. Kaneko ef al. (1959) have found normal PBI values together with other measurements indicative of hypothyroid states in dogs and Danowski et al. (1946) and Mayer ( 1947) have raised the question whether the dog is dependent on thyroid hormone production at all. Based on general histophysiological principles as summarized by Tonutti ( 1956), Tepperman ( 1962)) Stanbury and Murray ( 1962), and Rawson ( 1965), the mechanism of the pigment formation could be postulated as follows: The oral administration of propylthiouracil resulting in an inhibition of iodide peroxidase, decreased amounts of thyroid hormones in the peripheral circulation and increased release of thyroid-stimulating hormone (TSH) caused a high degree of morphologic activation and prevented the formation of pigment. The prevention of the minocycline-induced hyperplastic changes and pigmentation by the administration of thyroid powder can be considered as evidence that thyroid glands in an inactive storage phase with low enzymatic activity are not able to form dark pigment deposits. In summary, regardless of the morphologic changes provoked either by thy-
MORPHOLOGIC
EFFECTS
OF
MINOCYCLINE
167
roid powder (decreased rate of thyroxine synthesis antagonizing the action of TSH ) or by propylthiouracil (blocking of oxidizing enzymes), the gland is not synthesizing any substantial amounts of thyroid hormones. Both states, hyperplastic changes and extreme storage phase, are therefore indicative of decreased cellular function and it is reasonable to assume that in both instances the activity of oxidizing enzymes was diminished. The results of this experiment lend support to the theory that minocycline interferes with an iodide peroxidase in the thyroid gland and is itself degraded to a black nonfluorescent pigment presumably by an enzymatic reaction. Since no new uhrastructural entity was found in the folicular cells of minocycline-treated rats, the nature of this enzymatic pigmentation process must be explained on the basis of preexisting structures. In addition, the dark brown intracellular colloid droplets that were visible in PAS-stained preparations already suggested that the pigment was deposited within colloid droplets. The following observations lend support to the concept that the brown substance of the brown-black bodies as seen by electron microscopy is similar if not identical with normal colloid droplets: ( 1) The morphologic characteristics (size, shape, intracellular distribution, ultrastructural characteristics) are quite similar. (2) No evidence of foreign elements that might be construed as formative stages of these black bodies has been found. (3) No unusual deposits of pigments, dense aggregates, or other cell inclusions that might be identified as products of intracellular drug metabolism have been seen. Therefore, the intracellular pigment deposits are most probably precursors of colloid or fully developed colloid droplets which have either incorporated the drug itself or one or more metabolic products resulting in increased electron opacity. This process resulted in increased stainability (with osmium and lead) of these colloid structures as well as an increase in natural electron density as compared with similar elements in control material. According to Tonutti (1956) and Wetzel et al. (1965) oxidative enzyme systems are presumably located in the colloid droplets and originate in the vicinity of the Golgi complex. It is usually accepted that these colloid droplets are being discharged into the follicular lumina. The findings in dogs indicate that the large pigmented bodies in the colloid are probably coacervates of these small primary, pigmented colloid droplets. This process was not observed in rats since no intracolloid pigmentation was noticeable at the end of the 1 month experiment. However, all animals showed some pigment deposition in the colloid and in the epithelium after recovery. We therefore concluded that some of the pigmented colloid droplets were discharged into the follicular lumen while others remained within the cell. This seems to be in agreement with the generally accepted concept that colloid is formed by the cells and is secreted into the lumen where it is stored, whereas a minority (Nadler et al., 1962; van Heyningen, 1965) postulates that intracellular colloid originates from the follicular colloid. Dempsey (1962) and Wetzel et al. (1965) believe that both processes occur. Our findThe black bodies first appear in the Zweek ings support the first viewpoint. treated animals within the follicular epithelium, become abundant after 4 weeks
168
K.-F.
BENITZ,
G.
K.
S. ROBERTS,
AND
A.
YUSA
of treatment, and after the cessation of drug administration are discharged into the follicular lumen. The other possibility still remains open that pigmented colloid particles are taken up by the microvilli of the internal cell border and deposited again inside the epithelial cell. Therefore, an exchange of pigmented colloid material cannot be ruled out on the basis of our morphologic findings. It should be noted, however, that Kelly and Kanegis (1967) found substantial quantities of radioactivity in the thyroid glands of dogs that were treated with radioactive minocycline intravenously. Apparently this organ has a specific affinity for this compound. The presence of an unknown pigment in the epithelial cells of a vital organ such as the thyroid gland could be construed as a rather serious toxic effect. However, other clinically accepted and therapeutically useful tetracycline derivatives cause similar morphologic changes in laboratory animals. Deichmann et al. (1964) have recently shown that tetracycline and oxytetracycline cause the deposition of a brown pigment in the thyroid glands of rats when administered at dietary concentrations of O.Ol-0.3% from 3 to 9 months. Similar pigment deposition was found in the thyroid glands of dogs receiving tetracycline hydrochloride at dietary levels of O.l-1.0% from 12 to 24 months. Blackwood et al. (1966) claim that several of the new 6-methylene tetracyclines can also cause thyroid gland pigmentation after prolonged oral or parenteral administration to laboratory animals. Some of Deichmann’s findings are consistent with the results of earlier experiments carried out in 1956 in our laboratories. The thyroid glands of beagle dogs that received 200 or 400 mg/kg/day of tetracycline for 1 year showed macroscopically a dark discoloration associated with the microscopic presence of brownish yellow pigment. However, when tetracycline was given to rats at dietary concentrations of 0.02-0.5% for two years, no pigmentation was found. Even dietary levels of 5% tetracycline administered to rats for two to three years did not result in any morphologic changes in the thyroid gland. The discrepancy of these observations made in different laboratories cannot be explained at the present time. It should be noted that when tested in our laboratories, tetracycline, oxytetracycline, and chlortetracycline did not cause any Calesnick et al. (1954) reported an antithymorphologic activation. However, roid effect of chlortetracycline in rats; therefore minocycline does not seem to be unique in this respect. The data from our experiments suggest that the minocycline-induced pigmentation is an intracellular oxidation product. The fact that tetracycline derivatives caused pigmentation of thyroid glands of laboratory animals does not necessarily mean that it also happens in man. As a matter of fact, the publication of Deichmann et al. (1964) appeared at a time when many tetracycline derivatives had been in clinical use for a considerable number of years. According to our present knowledge the tetracycline-induced pigmentation of the thyroid gland in laboratory animals does not seem to carry over to man. AS of this date, we are not aware of any implications that tetracyclines are connected with thyroid gland disturbances, and a recent survey on the clinical side effects of tetracyclines published by Schindel (1965) fails to mention any of these effects.
MORPHOLOGIC
EFFECTS
OF
169
MINOCYCLINE
SUMMARY The morphologic effects of minocycline were studied in rats, dogs, monkeys, and mice using various routes of drug administration and graded doses. The results of these short-term studies ranging from 27 to 38 days can be summarized as follows: Minocycline caused various degrees of hemolytic anemia in dogs after the intravenous infusion of 10-40 mg/kg/day for 27-29 days, but not after 5 mg/kg/day. After one month, a black discoloration of the thyroid glands was found in monkeys (30 mg/kg/day; orally), dogs (40, 20, 10, and 5 mg/kg/day; intravenously), rats (75, 25, and 8 mg/kg/day; orally), but not in mice (250 mg/kg/day; orally). Rats and dogs showed a deposition of pigment in the thyroid glands sometimes associated with hyperplastic changes. In the canine species this hyperplasia was associated with the formation of thyroidectomy cells in the pituitary gland after the administration of 40 and 20 mg/kg/day of minocycline intravenously for 27-29 days. The pigmentation was much less pronounced in monkeys than in any other species. NO hyperplastic changes were noticeable. Electron microscopic observations and special staining procedures indicated that the nonfluorescent pigment was formed within the intraepithelial colloid droplets. The simultaneous administration of propylthiouracil plus minocycline or thyroid powder USP plus minocycline to rats caused morphologic activation or hyperplastic changes phase phenomena, respectively, without pigmentation of the thyroid glands. These findings indicate that oxidative enzyme activity (presumably absent in flat thyroid epithelium associated with the storage phase or blocked by propylthiouracil) is necessary for the deposition of pigment. The pigment was still present in thyroid glands of rats that received 75 mg/kg/day for 38 days and were then kept for one year on a normal diet. No other changes were seen at this time. The oral administration of minocycline caused yellow discoloration of femora and skulls without any deleterious effect on bone growth. ACKNOWLEDGMENT The authors gratefully acknowledge the technical assistance R&en, and Donald Dambach, Alfred W. Kramer, Jr., Roger assistance of Doris H. Lennon.
of Emil Semonick
Bohnel, and the
Gudrun editorial
REFERENCES BARKA, T., and ANDERSON, P. J. ( 1963). Histochemistfy: Theory, Practice and Bibliography, pp. 184-185. Harper (Hoeber), New York. BLACK~OOD, R. K., RENNHARD, H. H., BWEBOOM, J. J., and STEPHENS, C. R., JR. (1966). 6-Demethyl-6-halomethyl-5a,6-anhydrotetracyclines. U.S. Patent No. 3,264,348, August 2, 1966. CALESNICK, B., HARRIS, W. D., and JONES, R. S. ( 1954). Antithyroid action of antibiotics. Science 119, 128-129. COHLAN, S. Q., BEVELANDER, G., and BROSS, S. (1961). Effect of tetracycline on bone growth in the premature infant. Antimicrobial Agents and Chemotherapy. Proc. 1st Interscience Conf. Antimicrobial Agents and Chemotherapy, pp. 34&347. American Society of Microbiology, New York. COHLAN, S. Q., BEVELANDER, G., and TIAMSIC, T. (1963). Growth inhibition of prematures receiving tetracycline. Am. J. Diseases Children 105, 453-461. DANOWSKI, T. S., MAN, E. B., and WINKLER, A. W. (1946). Tolerance of normal, of thyroidectomized, and of thiourea or thiouracil treated dogs to oral desiccated thyroid and to intravenous thyroxine. Endocrinology 38, 230-237. DEICHMANN, W. B., BERNAL, E., ANDERSON, W. A. D., KEPLINGER, M., LANDEEN, K. and MCDONALD, W. (1964). The chronic oral toxicity of oxytetracycline HCl and tetracycline HCl in the rat. dog and pig. Ind. Med. Surg. 33, 787-806.
170
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G. K.
S. ROBERTS,
AND
A.
YUSA
DEMPSEY, E. W. (1962). Anatomy of the thyroid. In: The Thyroid (S. C. \Yerner, et{.), 2nd ed., pp. 113-114. Harper (Hoeber), New York. EKHOL~~, R. (1964). Thyroid gland. In: Electron Microscopic Anatomy (S. hl. Kurtz, ed.), Chapter 9, pp. 233-237. Academic Press, New York. HALLESY, D. W., and BENITZ, K.-F. ( 1963). Sulfobromophthalein sodium retention and morphological liver damage in dags. Toxicol. AppI. PharmacoZ. 5, 650460. HUECK, W. (1912). As quoted in Pearse, A. G. ( 1960), p. 925. KANEKO, J. J., TYL~, W. S., WINI), A., and CORNEL.IUS: C. E. ( 1959). Clinical applications of the thyroidal I131 uptake test in the dog. J. Am. Vet. Aled. Assoc. 135, 516-520. KELLY, R., and KANEGIS, L. A. ( 1967). Metabolism and tissue distribution of radioisotopically labeled minocycline. Toxicol. Appl. Pharmacol. 11, 171-183. LILLIE, R. D. ( 1948). Histopathologic Technique, pp. 143-145. Blakiston, Philadelphia, Pennsylvania. LILLIE, R. D. ( 1956). Nile blue staining techniqlle for the differentiation of melanin and lipofuscins. Stain TechnoZ. 31(4), 151. MALLORY, F. B. ( 1942). Pathological Technique, p. 138. Saunders, Philadelphia, Pennsylvania. MARTELL, M. J., JR., and BOOTHE, J. H. (1967). The 6-deoxytetracyclines. VII. Alkylated aminotetracyclines possessing unique antibacterial activity. J. Med. Chem. 10, 4446. MAYER, E. (1947). Inhibition of thyroid function in beagle puppies by propylthiouracil without disturbance of growth or health. Endocrinology 40, 165-181. NADLER, N. J., SARKAR, S. K., and LEBLOND, C. P. (1962). Origin of intracellular colloid droplets in the rat thyroicl. Endocrinology 71, 120-129. NOBLE, J. F., KANEGIS, L. A., and HALLESY, D. W. ( 1967). Short-term toxicity and observations on certain aspects of the pharmacology of a unique tetracycline-minocycline. ToxicoZ. AppZ. Pharmacol. 11, 128-149. PEAIISE, A. G. (1960). Histochemistry: Theoretical and Applied, 2nd ed., Appendix 23, pp. 920-926. Little, Brown, Boston, Massachusetts. POPPER, H. P., and SCHAFFNER, F. (1957). The Lioer: Structure and Function, p. 488. McGraw-Hill (Blakiston), New York. RAWSON, R. W. (1965). The thyroid gland. CZin. Symp. (CIBA) 17, l-63. REDIN, G. S. (1967). Antibiotic activity in mice of minocycline, a new tetracycline. Antimicrobial Agents Chemotherapy 1966. SCHINDEL, L. E. ( 1965). Clinical side effects of the tetracyclines. Antibiot. Chemotherapia 13, 300-316. STANBURY, J. B., and MURHAY, I. P. C. (1962). Synthesis, storage and release of thyroid hormone. In: The Thyroid (S. C. Werner, ed.). 2nd ed., pp. 10-25. Harper (Hoeber), New York. TEPPERICAN, J. (1962). Metabolic and Endocrine Physiology, pp. 89-92. Year Book Medical Publishers, Chicago, Illinois. TONUTTI, E. (1956). Normale Anatomic der endokrinen Driisen und endokrine Regulation. In: Lehrbzlch der speziellen pathologischen Anatomic (E. Kaufman, ed.), Vol. I, Part II, pp. 1287-1296. Walter De Gruyter, Berlin. VAN HEYNINGEP~‘, H. E. ( 1965). C orrelated light and electron microscope observations of glycoprotein-containing globules in the follicular cells of the thyroid gland of the rat. J. Histochem. Cytochem. 13, 286-295. WETZEL, B. K., SPICER, S. S., ancl WOLIAIAX. S. H. (1965). Changes in fine structure and acid phosphatase localization in rat thyroid cells following thyrotropin administration. J. Cell Biol. 25, 593-613. WILCOXO~, F. (1945). Individual comparisons by ranking methods. Biometrics 1, 80-83. WILCOXOX, F. ( 1947). Probability tables for individual comparisons by ranking methods. Biometrics 3, 119-122. WINTRORE, M. M. (1961). CZinicaZ Hematology: 5th ed., p. 519. Lea & Febiger, Philadelphia, Pennsylvania.
AND
APPLIED
Morphologic
PHAR.vIACOLOGY
11,
150-170
Effects of Minocycline
( 1967 )
in laboratory
Animals
K.-F. BENITZ, G. K. S. ROBERTS,AND A. YUSA Toxicology
Research Section, Lederle Laboratories Division, American Pearl River, New York 10965 Received April
Cyanamid
Compa1~~4,
10, 1967
The synthesis of minocycline, a new antibiotic of the tetracycline group, was reported by Martell and Boothe ( 1967). The compound is chemically defined as 7-dimethylamino-6-demethyl-6-deoxytetracycline. Redin ( 1967) has shown that this compound has unique and potentially useful antibacterial properties. The results of studies on drug metabolism and tissue distribution of this compound have been reported by Kelly and Kanegis ( 1967). Short-term toxicity experiments and observations of certain aspects of the pharmacology of minocycline have been reported by Noble et al. ( 1967). Our studies report the nature and the degree of morphologic effects of this drug in several species of laboratory animals after short-term administration of the drug. Long-term toxicity studies are in progress and the results will be reported at a later date. MATERIAL
AND
METHODS
Rats ( Sherman strain), dogs (purebred beagles), monkeys (Cercopitlrecus aetIriops), and mice (CF, albino) were used. At the end of each experiment the nonrodents were sacrificed by intravenous injection of sodium pentobarbital whereas chloroform inhalation was used for rodents. In two studies (One-Month Study in Rats and One-Month Study in Dogs) complete autopsies with inspection of all three body cavities and determinations of body weights and organ weights were performed. Paired organs were weighed together, and the weight of the thyroid gland was obtained after fixation. Representative samples of all major organs (heart, trachea. lungs, tongue, salivary glands, esophagus, stomach, small and large intestines, liver, gall bladder, pancreas, kidneys, urinary bladder, thymus, spleen, mesenteric lymph nodes, femoral marrow, pituitary gland, thyroid gland, adrenal gland, gonads, seminal vesicles and prostate, uterus, femur, skin, eyes, brain, and spinal column) were placed in suitable fixatives (4% aqueous formaldehyde, 8% neutral formaldehyde, and Bouin’s, Zenker’s? SUSA, and Helly’s solutions ), and paraffin sections from this material were used for a variety of conventional staining methods (hematoxylin and eosin, luxol-fast-blue-cresyl-fast-violet, Masson’s trichrome method, and iron reaction). In some instances unstained paraffin and cryostat sections were examined with a phase-contrast system. The following histochemical procedures were used for thyroid gland material: Turnbull’s reaction (Mallory, 1942), periodic acid-Schiff stain (Lillie, 1948), Nile blue stain ( Lillie, 1956), Nile bl ue stain (Hueck, 1912), Schmorl’s method for lipofuscins 150
MORPHOLOGIC
EFFECTS
OF
MINOCYCLINE
151
(Pearse, 1960), Ziehl-Neelsen stain (Pearse, 1960), ferrous iron reaction ( Barka and Anderson, 1963), methenamine silver method (Barka and Anderson, 1963), and bleaching with HaOz or peracetic acid (Pearse, 1960). Thyroid gland material of animals selected from the high dose groups of these rat and dog studies was examined by fluoresence microscopy. Similar techniques were used for additional studies; however, the morphologic examinations were limited to the thyroid glands. Samples for electron microscopy were removed quickly from the animal under light chloroform anesthesia and were fixed in cold (O-5” ) 1% osmium tetroxide in 0.1 M phosphate buffer pH 7.35 for 60 minutes, dehydrated, cleared in styrene, and embedded in Vestopal W. Additional samples were also fixed in cold (O-5” ) 5.0% glutaraldehyde in 0.1 M phosphate buffer pH 7.35 for 2 hours followed by 20 minutes in cold 1% osmium tetroxide, dehydrated in alcohol, cleared in propylene oxide and embedded in Maraglas-Cardolite. Some samples fixed in glutaraldehyde were embedded in Maraglas-Cardolite without postfixation in osmium tetroxide for comparison with other preparative procedures. Silver and light gold sections stained with lead citrate or uranyl acetate were examined using magnifications ranging from 3000 X to 30,000 X. Numerical data from all studies were examined for significant differences among groups using the rank test method of Wilcoxon ( 1945, 1947). Some morphologic changes were graded using as scores: absent, doubtful, slight, moderate, and marked. RESULTS
One-Month
Study in Rats
Eighty animals of both sexes were used for this study. Twenty rats served as controls whereas 3 other groups received 75, 25, and 8 mg/kg/day of minocycline by gavage for 31-35 days. Complete histologic examinations were carried out on the high dose group and the control animals. Observations during life have been reported by Noble et al. ( 1967). No morphologic changes were found that could be attributed to treatment except in the thyroid glands and the skeleton. The findings in the thyroid glands are summarized in Table 1 and illustrated in Fig. 1. It is obvious from these data that the oral administration of minocycline caused a marked black discoloration sometimes associated with hyperplastic changes (morphologic activation). This discoloration was dose related and was present in all treatment groups. A dark pigment was also seen histologically. Although some insignificant pigment granules were normally present in the glands of controls, the drug-induced pigmentation was much more pronounced, It was more conspicuous in follicles with high epithelium than in others that were lined with low cuboidal cells. It consisted of nonfluorescent dustlike particles (average diameter approx. l-2 p) of brown-black color (depending on the thickness of the sections) and was predominantly located in methods that the apical portion of the follicular epithelium. The histochemical were applied to some thyroid specimens gave inconclusive results. The Turnbull
(g):
(mg):
a Values in parentheses c Significantly different
IO/l0 -
IO/l0 -
lO/lO lO/lO -
lo/lo -
lo/lo -
TABLE
1
different
from
control
5/10 5/10
lo/lo
Y/10 7/10
7/10 3/10
l/l0 Q/l0
l/l0 Q/l0 -
Q/l0 l/l0
F 206 (186-232) 19.6 (17-23) 9.5 (7.8-11.0)
e/10 s/10
lO/lO -
6110 J/10
M 303 (23&349) 25.8C (20-35) 8.5c (6.6-10.8)
75 mg/kg/day
OBTAINED FROM MALE AND FEMALE (BABE) ORALLY FOR ONE MONTHQ
b Significantly I 6 0.5).
-
IO/l0 -
lo/lo -
-
lO/lO
F 216 (189-246) 19.1 (17-23) 8.9 (6.9-10.6)
GLANDS
lo/lo -
group(P
0 (Controls)
IN THYROID
M 284 (249-314) 20.9 (17-27) 7.3 (6.6-8.6)
FINDIN(;S
= ranges. from control
Relative weights (mg/ 100 g body weight):
weights
weight
Absolute
Sex: Body
Dose:
OF MORPHOLOGIC
Gross findings a. Size Normal Slightly enlarged b. Color Pink Black Dark brown Brown Red brown Dark red c. Isthmus Invisible Prominent Microscopic findings a. Hyperplastic changes Absent Slight b. Pigmentation Absent Slight Moderate Marked
SUMMARY
25 w/Wday
RECEIVING
group
(P
IO/10
5 0.01).
3 / 10 7/10
S/l0 7/1n
5110 5/10
Q/l0 l/10
M 3%‘b (W-385) 23.6 (a&29) 7.3 (5.2-8.7)
RATS
a/10 s/10 -
Q/l0 l/l0
3/10 7/10
4/10 l/IO 5/10 -
Q/10 l/10
F 207 (19&223) 18.4 (16-23) 8.9 (7.9-10.3)
VARI~C~
DOSE
OF MINOCYCLINE
R/l0 ?/IO !I/10 l/l0 -
8/10 e/10
7110 3/10
6jlO 4110
9110 l/10
2/10 S/l0
lo/lo
F 207 (181-231) 16.8 (12-22) 8.1 (7.3-10.0)
8 mdkgldw
3/10 2jlO 5/10
lo/lo
M 323b (264-371) 24.4 (19-31) 7.6 (6.1-9.1)
LEVELS
MORPHOLOGIC
FIG. 1. Morphologic male, 75 mg/kg/day fication: X 680.
EFFECTS
OF
153
MINOCYCLINE
activation with marked pigmentation of minocycline per OS for 35 days.
of thyroid gland. Rat Formalin, hematoxylin-eosin.
No.
K 147, Slagni-
reaction was negative whereas both Nile blue stains and the Schmorl procedure gave positive results as with lipofuscins. The Ziehl-Neelsen stain for acidfast lipofuscins was negative. Both methods for the detection of melanins, ferrous iron method and methenamine silver technique, were negative whereas exposure to Hz02 (48 hours) or peracetic acid (16 hours) resulted in bleaching. The PAS stain showed a reddish-brown reaction site, presumably a combination of black pigment with a red-stained polysaccharide component indicating that this material was of colloid nature. The periodic acid Schiff reaction gave a distinct brown color that was interpreted as a combination of black pigment and magenta colloid material of polysaccharide nature. The morphologic activation consisted of proliferation of the follicular epithelium associated with an increase in cell height, decrease in stainable colloid, and capillary hyperemia. These slight hyperplastic changes that were present in all treatment groups, as shown in Table 1, were not associated with any changes in serum protein-bound iodine values (Noble, et al., 1967). One could, therefore, conclude that the oral administration of minocycline did not cause any marked hypothyroidism in this species. A summary of the morphologic observations covering the changes in the skeletal system is given in Table 2. The measurements of the femur length indicate that the administration of minocycline did not result in any impairment of
54
K.-F.
BENITZ,
G.
IL
S. NOBERTS,
AND
A.
YUSA
MORPHOLOGIC
EFFECTS
OF
MINOCYCLINE
155
skeletal growth. This was confirmed by the microscopic examination of the distal epiphysis using various criteria such as the width of the epiphyseal disk, the arrangement of cartilage cells, the amount of trabecular bone, and the thickness of the cortex. Some of the femora from the high dose group were examined grossly under ultraviolet light (3660 A) and although they were distinctly yellow at autopsy, no fluorescence was seen. All other pathologic findings consisted of disease entities known to occur in rats, and no effects of treatment on incidence, nature, or degree of these spontaneous diseases were noticeable. One-Month
Study in Dogs
Ten beagle hounds were used and divided into 5 groups (1 male and 1 female per group). Doses of 40, 20, 10, and 5 mg/kg/day of minocycline were given by the intravenous route for 27-29 days. The control animals received saline. The results of observations during life have been reported by Noble et al. (1967). The intravenous administration of minocycline caused the following toxic effects: (1) morphologic activation and pigmentation of the thyroid gland together with formation of thyroidectomy cells in the pituitary gland and (2) siderotic changes consistent with and attributable to intravascular hemolysis. In the two animals receiving the highest dose (40 mg/kg/day) these changes were accompanied by the formation of occasional bile plugs. The findings in the thyroid gland together with the changes in the pituitary gland are given in Table 3. The nonfluorescent pigment was grossly and microscopically not clearly dose dependent. As shown in Fig. 2 in some instances larger clumps of presumably the same pigment were present in the colloid. The morphologic activation was present in 3 of 4 animals of the two high-dose groups. This was mediated by the pituitary gland as evidenced by the presence of thyroidectomy cells. The amount of hemosiderin in spleens, livers, and kidneys of the animals receiving 40 or 20 mg/kg/day of minocycline was presumably attributable to intravascular hemolysis (see Table 4). According to Noble et ~2. ( 1967) a dose of 10 mg/kg/day caused only very slight hematologic changes that were almost within the range of normal fluctuation. However, in view of the hematologic findings in the animals of the 40 and 20 mg/kg/day groups, these changes could be considered as suggestive of a slight degree (trace) of hemolytic anemia although they were not accompanied by any abnormal siderotic changes except for a moderate increase in iron-positive pigment in the spleen. The presence of occasional bile plugs does not necessarily indicate hepatic (elevated sulfobromophthalein retencell damage since, with one exception tion), all liver function tests were within normal ranges (Noble et al., 1967). In addition, no morphologic changes indicative of hepatocellular damage were evident. The drug-induced erythema as described by Noble et al. (1967) was not found at autopsy or in microscopic preparations. This is not surprising since
body
FINDIKGS
also in colloid.
M F M F M F M F M F
Sex
OF MOHPHOLOQIC
0 Pigmentation
5
10
PO
40
0
Dose (Wkiday)
SVMMARY
7.3
9.7
7.2
8.2
6.3
1.3 0.7
0.9 1.2 0 6 0.8 0.9
6.5
7.2 9.5
(6’) 0.5 0.6 0.6
-
weight
.4bsolute
GLAXDS
8.1 9.4
Final weight (kg) -
IX THYROID
TABLE
3
findings
96
134
125
95 98
125 126
9%
62 64
Relative weight (w/k)
Gross
Pale pink Pale pink Black Black Black Black Black Black Gray Black
Color at autopsy Absent Absent Marked Absent Moderate Marked Moderate Moderate Slight Slight
Hyperplasia
Microscopic
OBTAINED FHOM MALE AND FEMALE Dots RECEIVING (Rasp) INTHAVENOUSLY FOR ONE MONTH
DOSE
Absent Absent Marked” Slight Moderate Moderate” Slight Slight Slight Moderatea
Pigmentation
findings
V4~ror-s
Absent Absent Present ilbsent Present Present hbsent Absent dbsent .4bsent
Thyroidectomy cells in pituitary gland
T,EVCLS OF MIXWYPLINE
MORPHOLOGIC
erythema belongs to the transient appear immediately after death. One-Month
Study
EFFECTS
capillary
OF
157
MINOCYCLINE
phenomena
that are known
to dis-
in Monkeys
It was, of course, of interest to determine whether these thyroid gland changes could also be found in other species. Since both the rat and the dog reacted with morphologic activation and pigmentation, an additional experiment in African green monkeys was carried out to determine whether pig-
FIG. apical colloid.
2. Marked morphologic portion of follicular Dog P 4494, male,
activation of thyroid gland epithelium and heavy pigment 40 mg/kg/day of minocycline
with fine granular pigmentation in deposits within barely stainable intravenously for 30 days.
mentation occurred and was associated with hyperplastic changes. Observations during life have been reported by Noble et al. ( 1967). Three male and 3 female monkeys received 30 mg/kg/day of minocycline per OS, in gelatin capsules for 30 days. Two female animals served as controls. All animals survived the experimental period with the exception of 1 male that died spontaneously after receiving 22 doses. The cause of death was not determined. The morphologic examination was limited to the thyroid glands. The results are given in Table 5. These data indicate that monkeys reacted slightly differently from rats and dogs. Although all thyroid glands of the treated group were grossly discolored, only one half of the animals showed pigmentation microscopically. There was no morphologic activation associated with these
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changes. In general, the degree of pigmentation was much less than in the dog, the other nonrodent species that could be used for comparison. One could speculate that the lack of an active hyperplastic process could have been responsible for the inconsistent reactivity pattern with respect to pigment deposition.
of
Mode
Action
Study
in Rats
These experiments showed that minocycline caused hyperplastic changes and dark pigmentation in the thyroid glands of various species. Many antithyroid drugs, for instance the thioureas, are believed to act by inhibiting a peroxidase system necessary for the oxidation of inorganic iodine prior to iodination of tyrosine. This mechanism of action and the observations made by Kelly and Kanegis (1967) that minocycline is easily oxidized to an insoluble black substance suggested that it might be a competitive inhibitor of iodine peroxidase, being oxidized to a black pigment in the process. If this were the case, one might expect an antithyroid compound such as propylthiouracil to inhibit the peroxidase system and therefore to prevent the oxidation of minocycline and thus the T.4BLE SUMMARY BILE
OF DEGREES
PLUGS
OF ANEMIA,
OBSERVED
IN
ORGAN
4
SIDEROSES,
DOGS RECEIVIKG TNTRAVENOT-SLY
AND
PRESENCE
Siderosis Dose (mg/kg/day)
Sex
0
M F M F M F
40 20
F
Absent Absent Moderate Moderate Slight Slight Trace Trace
details,
see Noble
M
10
(1 For
Degree of anemia”
hematologic
Slight Slight Marked Marked Moderate Moderate Moderate Moderate
SUMMARY
OF MORPHOLOGIC! RECEIVING
FIXDINGS
30 MG/KG/DAY
of Kidney
Slight Slight AMarked Marked Slight Moderate Slight Slight
Absent Slight Marked Moderate Absent Slight Absent Slight
Controls Treated
Gross
GL~XDS
OBTAINED
ORALLY OF MINOCYCLINE ITPITH CONTROLS
IN THYROID
(BASE)
findings
Normal pink with invisible isthmus Dark red-brown Brown Black 5/6 Prominent isthmus Invisible isthmus
Hepatic hile plugs Absent Absent Present Present Absent .4bsent ;\hsent
5 FROM
Hyperplasia
6 MALE
FOR 30 Ds~s
Microscopic Group
(BOSE)
(1967).
TABLE MONKEYS
OF HFPATI~
OF MINOCYCLINE
Liver
Spleen
et al.
OR ABSENCE
V.~RIOUS DOSE LEVELS FOR ONE MONTH
.\s\‘u
FEMALE
IN COMPARISON
findings Pigmentation
Absent
?ja
Absent
2b
Absent
616
Doubtful
3/6
Slight Moderate
‘3/6 l/6
%ja
I/‘3 5/6 l/6
MORPHOLOGIC
EFFECTS
OF
MINOCYCLINE
159
pigment deposition. The same inhibitory process should still cause a morphologic activation of the gland. Conversely, if the enzymatic activity of the gland could be suppressed by an exogenous thyroid substance, one could expect the prevention of the minocycline-induced morphologic activation and also the pigmentation of the gland. This hypothesis was tested in the following experiment. Eighteen male Sherman rats (4 or 5 per group) were treated for 1 month. One group served as controls. A second group received minocycline at a standard dose of 100 mg/ kg/day for 5 days per week by gavage. The third group received the standard dose of minocycline and a 1.0% drug diet of thyroid powder USP (average intake approximately 800 mg/kg/day ), Propylthiouracil USP was given to a fourth group in drinking water as an 0.01% solution (average intake approximately 8 mg/kg/day) along with the standard dose of minocycline. The results of this experiment are given in Table 6. Again, minocycline alone caused a deposition of pigment and moderate morphologic activation of the thyroid glands in all animals. However, the simultaneous administration of minocycline plus propylthiouracil resulted in a marked degree of hyperplastic changes without any pigmentation. These observations suggest that thyroid glands undergoing rapid hyperplastic and hypertrophic changes were not able to form any pigment presumably derived from minocycline. The administration of minocycline plus thyroid powder resulted in an extreme storage phase and again no pigmentation was found. Since the administration of thyroid powder obviated the need for an active synthesis of thyroid hormones, this hypotrophic state of activity also interfered with the formation of minocycline-induced pigmentation. These results confirm the statement that minocycline has an antithyroid effect and support the speculation that enzyme activity in the gland is necessary for the formation of the pigment. The most likely system seems to be an iodide peroxidase, and minocycline itself is the likely source of the pigment. Onset and Recovery
Study
in Rats and Mice
Preliminary experiments indicated that the pigmentation of thyroid glands occurs rather early, especially in rats. It could be tentatively identified 2 weeks after oral administration of 100-200 mg/kg/day. It was therefore of interest to study the early phases of pigmentation using electron microscopy. Fifteen adult rats were divided into 3 groups (5 animals per group). Three rats received 100 mg/kg/day of minocycline intraperitoneally; 2 animals served as controls. These groups were sacrificed at weekly intervals (after 1, 2, and 4 weeks of drug administration). In addition 2 animals received 144 mg/kg/day of minocycline orally and were sacrificed after 26 days of treatment. After 1 week of treatment, no ultrastructural differences between control and experimental animals were found. After 2 weeks of treatment a considerable number of black bodies was found in approximately 50% of the follicular cells. This change became more pronounced after 26-28 days of treatment and was usually confined to the apical cell region (Figs. 3 and 4). These structures were usually spherical or oval (approximately 0.1-1.5 p in diameter) and
0 Values in parentheses b In drug diet. c In drinking water.
4
Minocycline + 1.0% thyroid powderb Nnocycline + 0.01 y0 propylthiouracilC
= ranges.
5
4
Number of rats
ALONE
Knocycline
MONTH
&VDISGS
5
Treatment
ONE
OF MORPHOLOGIC
Controls
SUMMARY
THYROID
282 (26+316) 287 (266-306) 271 (26CH86) 238 (214-264)
k)
Body weight
OR IN COSJZ~NCTION
IS WITH
GLANDS
(77YO2)
(16::P)
(25Z3)
(266)
(md
Absolute weight
THYROID
OBTAINED POWDER
FROM
T$BLE
6 RECEIVIKG
findings
8.3 (7.6-9.7) 9.9 (8.2-10.9) 7.4 (7.0-7.7) 38.1 (29.2-44.4)
Relative weight (mg/lOO g B.W.)
Gross
100
Red
Pink
Black
Pink
515
4/J
414
515
Microscopic
5/5 Moderate hyperplasia 4/P Extreme storage phase 414 Marked hyperplasia 515
Normal
(BASE) CONTROLS
AMorphologic state
WITH
OF MISOCYCLIKE
IN COMPARISON
MG/KG/D.IY
Color at autopsy
_____
OR PROPYLTHIOUR.~CIL
RATS
.4hsent
Bbsent
Present
absent
Pigmentation
findings
ORALLY
FOH
MORPHOLOGIC
EFFECTS
OF
MINOCYCLINE
161
FIG. 3. Numerous electron dense bodies (DB) of varying dimensions, some of which show aggregates of darkly stained particles. A large, rather homogeneous colloid droplet (CD) of lighter density is present. M = rather prominent mitochondria; ER = ergastoplasm; h4V I microvilli; L = lumen. Thyroid gland of rat No. B 3906, female, 144 mg/kg/day of minocycline orally for 26 days. Five percent glutaraldehyde followed by 1.0% osmium-tetroxide. Magnification: X 19,950.
showed a homogeneous dark grayish black opacity after glutaraldehyde fixation (see Fig. 5). Postfixation with osmium followed by lead staining or lead staining alone after glutaraldehyde fixation resulted in the appearance of intense grayish black to dark black bodies as shown in Fig. 6. They consisted of a homogeneous matrix sometimes containing fine fibrous or threadlike elements,
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FIG. 4. Thyroid epithelium with nmI,erous small colloid droplets (CD) of medium density are dispersed among the ergastoplosm ( EH) containing dilated cisternae. N = nucleus: AIV = microvilli; L = lumen. Thyroid gland of rat No. B 3910, female, control. Five percent glutaraldehyde followed 1))~ 1.0% osmium-tetroxide. Magnification: x 19,950.
ringlike structures about 0.05 p in diameter with a light center and a dark rim, and some dense particles of various dimensions. These bodies appeared to have a membrane, and in some sections a thin tenuous line delineated these structure from the surrounding cytoplasmic matrix. The mitochondria were somewhat more pronounced than normal. Nuclei, endoplasmic reticulum, and Golgi entities were found complex did not show any alterations. No novel structural in the follicular epithehum. The morphologic characteristics of the dark bodies with respect to size, shape,
MORPHOLOGIC
FIG. without bodies female,
EFFECTS
OF
MINOCYCLINE
163
S. Electron dense bodies obtained from a thyroid specimen fixed in glutaraldehyde postosmification or heavy metal staining. Please note the natural opacity of these as well as the network of dense material within these aggregates. Rat No, B 390’7, 144 mg/kg/day of minocyclinc orally for 26 days. Magnification: x 51,3011.
Frr.. 6. Dense bodies containing typical ringlike elements. Thin membranes (MB) were found in close apposition to these bodies. Thyroid gland of rat No. B 3906, female, 144 mg/kg/day of minocycline orally for 26 days. Five percent glutnraldehyde followed by 1.0% osmilun tctroxide and stained with uranyl acetate and lead citrate. Magnification: x ,51,500.
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and distribution did not differ from the structures believed to be precursors of colloid droplets themselves except for their increased opacity and stainability. All other alterations were consistent with changes found in stimulated thyroid glands as described by Ekholm ( 1964). A similar time-sequence study was carried out in mice. Twenty animals were used for this study and 250 mg/kg of minocycline was administered orally for 28 days. Two treated and 2 control animals were sacrificed on days 3, 7, 14, 21, and 28 of treatment. The morphologic examination was limited to gross inspection which proved to be sufficient for the detection of pigmentation in other species. The neck regions of the animals were placed under a dissecting scope and the thyroid glands were exposed and inspected in situ for changes in size and color. Then the glands were removed together with the trachea and larynx and reexamined at a higher magnification. No difference in color or size between control and experimental animals was found although a rather high dose was used. These findings were in contrast with the observations made in rats, dogs, and monkeys, Except for possible species differences, this discrepancy cannot be explained at the present time although good absorption by mice was obtained in similar experiments ( Redin, 1967). The possible regression of pigmentation and hyperplastic changes was studied in 27 rats that received 75 mg/kg of minocycline for 38 days and were then kept for 1 year on a normal diet. An equal number of animals were kept as controls. The morphologic examination was limited to the thyroid gland. The results of this study are given in Table 7. Gross and microscopic findings indicated that the pigmentation was still present after 1 year of recovery. The slightly increased pigmentation in the control group in comparison with similar findings from the 1 month experiment was not surprising. These rats were about 14 months old, and it is known that the thyroid gland shows increased amount of pigmentation with age. Differences in microscopic appearance also helped to distinguish between treated and controls. The pathologic pigment usually consisted of coarse granules whereas the follicular epithelium of the control animals contained considerably fewer granules of smaller particle size. In all treated animals some pigment deposits were also found in the colloid, usually located next to the epithelial border. Colloid storage and follicular pattern were of the same degree and incidence in both treated and control groups. No signs of morphologic activation were found. One can therefore conclude that the pigment itself is probably not the cause of the hyperplasia of the thyroid gland as observed at the end of the l-month experiment. The presence of this unknown pigment did not change the gross and histologic characteristics of the glands. One can assume that once this pigment is deposited within epithelium and colloid, it remains there for a long time. However, the morphologic findings point to the fact that the presence of this foreign substance is apparently innocuous. DISCUSSION
The hemolytic anemia observed in dogs was rather mild and did not result in severe hemolytic crises, nor was it associated with jaundice. According to
MORPHOLOGIC
EFFECTS
TABLE ~I!MMARY RE~EIVED'~~
OF
&~ORPHOLOGIC MG/KG/DAYOF
FIKDINGS IN THYROID MIKOCYCLINE(B.ME)ORALLY
PERIOD
IN COMPARISON
OF
165
MINOCYCLINE
7 GL.4NDS OBT.4INED FHOM R.j'rs HAVING FOLLOWED BY A ONE-YE.W RECOVERY WITH
CONTROLP
Treated
Controls
Absolute
weights
(ma):
Cross findings a. Size Normal Questionably enlarged 1. Color Pink Black Brown-black Dark brown Dark pink Microscopic findings a. Colloid storage Absent Slight Moderate Marked b. Follicular pattern* Macro Mixed Micro c. Pigmentation’ Absent Slight Moderate Marked
Males 29.3 (25-35)
Females 22.1 (19-25)
lY/lS -
14114
11/13 -
14/14
AMales si.5 (41-36)
Females 25.0 (14-35)
13/13 -
13/14 l/IS -
lo/l3 Y/13 -
Y/13
7114 3/14 4114
2114
-
-
4113 6/13 3/13
6114 6114
4113 6/13
“2114
Y/l3
l/14 S/l4 7114 l/14
S/I3 7113 l/l3
8114 S/l4 -
o/13 Y/l3 2113
7114 6114 l/14
8114 5114 l/l4
-
u Values in parentheses = ranges. b Macro = predominantly macrofollicular terns interspersed; micro = predominantly c Based on phase contrast microscopy.
S/l3 l/l3 l/l3
pattern; mixed microfollicular
l/l4 4114 9/14
4/13 9113
= macro.pattern.
and
microfollicular
pat-
Noble et al. (1967) there was no visible evidence of drug-induced hemoglobinemia or hemoglobinuria. Usually, a hemolytic process of a subacute nature does not impair hepatic function (Popper and Schaffner, 1957). However, the increased sulfobromophthalein sodium retention observed in the two animals receiving 40 mg/kg/d ay was considered by Noble et al. (1967) as indicative of early hepatocellular malfunction. However, no morphologic evidence was found to substantiate this interpretation. The intrahepatic cholestasis may account for this functional change since according to Hallesy and Benitz (1963) hepatic siderosis per se does not cause abnormal BSP retention values. A dose of 5 mg/kg/day did not cause any hemolytic anemia nor did it cause any abnormal deposition of iron-positive pigment in spleen, liver, and kidneys.
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Therefore, these animals were excluded from Table 4. According to Wintrobe ( 1961) an anemia of similar type that was not accompanied by active regeneration of red cells has been found in cases of spontaneous hyperthyroid&m and also after the total ablation of the thyroid gland in man and laboratory animals. The yellow discoloration observed in the femur and calvarium of all treated rats was not surprising because it is well known that tetracycline derivatives are rapidly deposited in bony structures. Some of the bones were examined under ultraviolet light but did not show a yellow fluorescence in spite of the fact that they appeared yellow in ordinary light. Apparently the calcium chelate complex of minocycline did not fluoresce but Kelly and Kanegis (1967) have shown that magnesium forms a fluorophore with this compound. However, the femur length and microscopic examination of the femora from the 75 mg/kg/ day group did not show any difference from controls. These findings suggest that minocycline does not cause an impairment of postnatal skeletal growth, a phenomenon claimed by Cohlan et al. ( 1961, 1963) for tetracycline but unconfirmed by observations made in our laboratories. If any harmful effects had taken place one would have observed an effect in these rodents since the experiment was started 1 week after weanling age and continued during a period of rapid skeletal growth. The most striking drug effect was the abnormal pigmentation of the thyroid gland that was observed in rats, dogs, and monkeys. The rat and the dog reacted also with a hyperplastic process which was not detectable in monkeys. The hypothyroid condition of dogs treated with minocycline could already be suspected in viva since according to Noble et al. (1967) the serum proteinbound iodine (PBI) values decreased to 3042% of the predose values. However, in one of the high dose animals the morphologic activation was absent and no thyroidectomy cells were found in the pituitary gland. This inconsistency was probably not surprising since the usefulness of the PBI determinations in dogs is stil1 to be determined. Kaneko ef al. (1959) have found normal PBI values together with other measurements indicative of hypothyroid states in dogs and Danowski et al. (1946) and Mayer ( 1947) have raised the question whether the dog is dependent on thyroid hormone production at all. Based on general histophysiological principles as summarized by Tonutti ( 1956), Tepperman ( 1962)) Stanbury and Murray ( 1962), and Rawson ( 1965), the mechanism of the pigment formation could be postulated as follows: The oral administration of propylthiouracil resulting in an inhibition of iodide peroxidase, decreased amounts of thyroid hormones in the peripheral circulation and increased release of thyroid-stimulating hormone (TSH) caused a high degree of morphologic activation and prevented the formation of pigment. The prevention of the minocycline-induced hyperplastic changes and pigmentation by the administration of thyroid powder can be considered as evidence that thyroid glands in an inactive storage phase with low enzymatic activity are not able to form dark pigment deposits. In summary, regardless of the morphologic changes provoked either by thy-
MORPHOLOGIC
EFFECTS
OF
MINOCYCLINE
167
roid powder (decreased rate of thyroxine synthesis antagonizing the action of TSH ) or by propylthiouracil (blocking of oxidizing enzymes), the gland is not synthesizing any substantial amounts of thyroid hormones. Both states, hyperplastic changes and extreme storage phase, are therefore indicative of decreased cellular function and it is reasonable to assume that in both instances the activity of oxidizing enzymes was diminished. The results of this experiment lend support to the theory that minocycline interferes with an iodide peroxidase in the thyroid gland and is itself degraded to a black nonfluorescent pigment presumably by an enzymatic reaction. Since no new uhrastructural entity was found in the folicular cells of minocycline-treated rats, the nature of this enzymatic pigmentation process must be explained on the basis of preexisting structures. In addition, the dark brown intracellular colloid droplets that were visible in PAS-stained preparations already suggested that the pigment was deposited within colloid droplets. The following observations lend support to the concept that the brown substance of the brown-black bodies as seen by electron microscopy is similar if not identical with normal colloid droplets: ( 1) The morphologic characteristics (size, shape, intracellular distribution, ultrastructural characteristics) are quite similar. (2) No evidence of foreign elements that might be construed as formative stages of these black bodies has been found. (3) No unusual deposits of pigments, dense aggregates, or other cell inclusions that might be identified as products of intracellular drug metabolism have been seen. Therefore, the intracellular pigment deposits are most probably precursors of colloid or fully developed colloid droplets which have either incorporated the drug itself or one or more metabolic products resulting in increased electron opacity. This process resulted in increased stainability (with osmium and lead) of these colloid structures as well as an increase in natural electron density as compared with similar elements in control material. According to Tonutti (1956) and Wetzel et al. (1965) oxidative enzyme systems are presumably located in the colloid droplets and originate in the vicinity of the Golgi complex. It is usually accepted that these colloid droplets are being discharged into the follicular lumina. The findings in dogs indicate that the large pigmented bodies in the colloid are probably coacervates of these small primary, pigmented colloid droplets. This process was not observed in rats since no intracolloid pigmentation was noticeable at the end of the 1 month experiment. However, all animals showed some pigment deposition in the colloid and in the epithelium after recovery. We therefore concluded that some of the pigmented colloid droplets were discharged into the follicular lumen while others remained within the cell. This seems to be in agreement with the generally accepted concept that colloid is formed by the cells and is secreted into the lumen where it is stored, whereas a minority (Nadler et al., 1962; van Heyningen, 1965) postulates that intracellular colloid originates from the follicular colloid. Dempsey (1962) and Wetzel et al. (1965) believe that both processes occur. Our findThe black bodies first appear in the Zweek ings support the first viewpoint. treated animals within the follicular epithelium, become abundant after 4 weeks
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of treatment, and after the cessation of drug administration are discharged into the follicular lumen. The other possibility still remains open that pigmented colloid particles are taken up by the microvilli of the internal cell border and deposited again inside the epithelial cell. Therefore, an exchange of pigmented colloid material cannot be ruled out on the basis of our morphologic findings. It should be noted, however, that Kelly and Kanegis (1967) found substantial quantities of radioactivity in the thyroid glands of dogs that were treated with radioactive minocycline intravenously. Apparently this organ has a specific affinity for this compound. The presence of an unknown pigment in the epithelial cells of a vital organ such as the thyroid gland could be construed as a rather serious toxic effect. However, other clinically accepted and therapeutically useful tetracycline derivatives cause similar morphologic changes in laboratory animals. Deichmann et al. (1964) have recently shown that tetracycline and oxytetracycline cause the deposition of a brown pigment in the thyroid glands of rats when administered at dietary concentrations of O.Ol-0.3% from 3 to 9 months. Similar pigment deposition was found in the thyroid glands of dogs receiving tetracycline hydrochloride at dietary levels of O.l-1.0% from 12 to 24 months. Blackwood et al. (1966) claim that several of the new 6-methylene tetracyclines can also cause thyroid gland pigmentation after prolonged oral or parenteral administration to laboratory animals. Some of Deichmann’s findings are consistent with the results of earlier experiments carried out in 1956 in our laboratories. The thyroid glands of beagle dogs that received 200 or 400 mg/kg/day of tetracycline for 1 year showed macroscopically a dark discoloration associated with the microscopic presence of brownish yellow pigment. However, when tetracycline was given to rats at dietary concentrations of 0.02-0.5% for two years, no pigmentation was found. Even dietary levels of 5% tetracycline administered to rats for two to three years did not result in any morphologic changes in the thyroid gland. The discrepancy of these observations made in different laboratories cannot be explained at the present time. It should be noted that when tested in our laboratories, tetracycline, oxytetracycline, and chlortetracycline did not cause any Calesnick et al. (1954) reported an antithymorphologic activation. However, roid effect of chlortetracycline in rats; therefore minocycline does not seem to be unique in this respect. The data from our experiments suggest that the minocycline-induced pigmentation is an intracellular oxidation product. The fact that tetracycline derivatives caused pigmentation of thyroid glands of laboratory animals does not necessarily mean that it also happens in man. As a matter of fact, the publication of Deichmann et al. (1964) appeared at a time when many tetracycline derivatives had been in clinical use for a considerable number of years. According to our present knowledge the tetracycline-induced pigmentation of the thyroid gland in laboratory animals does not seem to carry over to man. AS of this date, we are not aware of any implications that tetracyclines are connected with thyroid gland disturbances, and a recent survey on the clinical side effects of tetracyclines published by Schindel (1965) fails to mention any of these effects.
MORPHOLOGIC
EFFECTS
OF
169
MINOCYCLINE
SUMMARY The morphologic effects of minocycline were studied in rats, dogs, monkeys, and mice using various routes of drug administration and graded doses. The results of these short-term studies ranging from 27 to 38 days can be summarized as follows: Minocycline caused various degrees of hemolytic anemia in dogs after the intravenous infusion of 10-40 mg/kg/day for 27-29 days, but not after 5 mg/kg/day. After one month, a black discoloration of the thyroid glands was found in monkeys (30 mg/kg/day; orally), dogs (40, 20, 10, and 5 mg/kg/day; intravenously), rats (75, 25, and 8 mg/kg/day; orally), but not in mice (250 mg/kg/day; orally). Rats and dogs showed a deposition of pigment in the thyroid glands sometimes associated with hyperplastic changes. In the canine species this hyperplasia was associated with the formation of thyroidectomy cells in the pituitary gland after the administration of 40 and 20 mg/kg/day of minocycline intravenously for 27-29 days. The pigmentation was much less pronounced in monkeys than in any other species. NO hyperplastic changes were noticeable. Electron microscopic observations and special staining procedures indicated that the nonfluorescent pigment was formed within the intraepithelial colloid droplets. The simultaneous administration of propylthiouracil plus minocycline or thyroid powder USP plus minocycline to rats caused morphologic activation or hyperplastic changes phase phenomena, respectively, without pigmentation of the thyroid glands. These findings indicate that oxidative enzyme activity (presumably absent in flat thyroid epithelium associated with the storage phase or blocked by propylthiouracil) is necessary for the deposition of pigment. The pigment was still present in thyroid glands of rats that received 75 mg/kg/day for 38 days and were then kept for one year on a normal diet. No other changes were seen at this time. The oral administration of minocycline caused yellow discoloration of femora and skulls without any deleterious effect on bone growth. ACKNOWLEDGMENT The authors gratefully acknowledge the technical assistance R&en, and Donald Dambach, Alfred W. Kramer, Jr., Roger assistance of Doris H. Lennon.
of Emil Semonick
Bohnel, and the
Gudrun editorial
REFERENCES BARKA, T., and ANDERSON, P. J. ( 1963). Histochemistfy: Theory, Practice and Bibliography, pp. 184-185. Harper (Hoeber), New York. BLACK~OOD, R. K., RENNHARD, H. H., BWEBOOM, J. J., and STEPHENS, C. R., JR. (1966). 6-Demethyl-6-halomethyl-5a,6-anhydrotetracyclines. U.S. Patent No. 3,264,348, August 2, 1966. CALESNICK, B., HARRIS, W. D., and JONES, R. S. ( 1954). Antithyroid action of antibiotics. Science 119, 128-129. COHLAN, S. Q., BEVELANDER, G., and BROSS, S. (1961). Effect of tetracycline on bone growth in the premature infant. Antimicrobial Agents and Chemotherapy. Proc. 1st Interscience Conf. Antimicrobial Agents and Chemotherapy, pp. 34&347. American Society of Microbiology, New York. COHLAN, S. Q., BEVELANDER, G., and TIAMSIC, T. (1963). Growth inhibition of prematures receiving tetracycline. Am. J. Diseases Children 105, 453-461. DANOWSKI, T. S., MAN, E. B., and WINKLER, A. W. (1946). Tolerance of normal, of thyroidectomized, and of thiourea or thiouracil treated dogs to oral desiccated thyroid and to intravenous thyroxine. Endocrinology 38, 230-237. DEICHMANN, W. B., BERNAL, E., ANDERSON, W. A. D., KEPLINGER, M., LANDEEN, K. and MCDONALD, W. (1964). The chronic oral toxicity of oxytetracycline HCl and tetracycline HCl in the rat. dog and pig. Ind. Med. Surg. 33, 787-806.
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