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2005 33: 517 Toxicol Pathol
Katsuhiko Yoshizawa, Grace E. Kissling, Jo Anne Johnson, Natasha P. Clayton, Norris D. Flagler and Abraham Nyska
Chemical-Induced Atrial Thrombosis in NTP Rodent Studies
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Toxicologic Pathology, 33:517–532, 2005
Copyright C ?by the Society of Toxicologic Pathology
ISSN: 0192-6233 print / 1533-1601 online
Chemical-Induced Atrial Thrombosis in NTP Rodent Studies
KATSUHIKO YOSHIZAWA,1GRACE E. KISSLING,2JO ANNE JOHNSON,1NATASHA P. CLAYTON,1NORRIS D. FLAGLER,1
AND ABRAHAM NYSKA1
1Laboratory of Experimental Pathology and2Biostatistics Branch, National Institute of Environmental Health Sciences (NIEHS),
Research Triangle Park, North Carolina 27709, USA
Cardiac thrombosis, one of the causes of sudden death throughout the world, plays a principal role in several cardiovascular diseases, such
as myocardial infarction and stroke in humans. Data from studies of induction of chemical thrombosis in rodents help to identify substances in our
chemical exposures and compared it to similarly induced lesions reported in the literature. Spontaneous rates of cardiac thrombosis were determined
for control Fischer 344 rats and B6C3F1 mice: 0% in rats and mice in 90-day studies and, in 2-year studies, 0.7% in both genders of mice, 4% in male
rats, and 1% in female rats. Incidences of atrial thrombosis were increased in high-dosed groups involving 13 compounds (incidence rate: 20–100%):
2-butoxyethanol, C.I. Direct Blue 15, bis(2-chloroethoxy)methane, diazoaminobenzene, diethanolamine, 3,3?-dimethoxybenzidine dihydrochloride,
hexachloroethane, isobutene, methyleugenol, oxazepam, C.I. Pigment Red 23, C.I. Acid Red 114, and 4,4?-thiobis(6-t-butyl-m-cresol). The main
localization of spontaneously occurring and chemically induced thromboses occurred in the left atrium. The literature survey suggested that chemical-
induced atrial thrombosis might be closely related to myocardial injury, endothelial injury, circulatory stasis, hypercoagulability, and impaired atrial
mechanical activity, such as atrial fibrillation, which could cause stasis of blood within the left atrial appendage, contributing to left atrial thrombosis.
Supplementary data referenced in this paper are not printed in this issue of Toxicologic Pathology. They are available as downloadable files at
http:taylorandfrancis.metapress.com/openurl.asp?genre=journal&issn=0192-6233. To access them, click on the issue link for 33(5), then select this
or a member subscription accessed through www.toxpath.org.
Heart; left atrium; atrial thrombosis; F344 rats; B6C3F1 mice; chemical-induced; NTP studies.
Thrombosis constitutes a serious disorder that can prove
fatal in humans with classical risk factors associated with
myocardial infarction, one of the most important of which is
becomes significant, for example, in aged women undergo-
involved in the induction of thrombosis (Rosendaal et al.,
2002; Lowe, 2004). Mitral valve disease, atrial fibrillation,
tal Pathology, NIEHS, Research Triangle Park, NC 27709, USA; e-mail:
Abbreviations: ATSDR: Agency for Toxic Substances and Disease
zene; DEL: diethanolamine; DMOB: 3,3?-dimethoxybenzidine dihy-
drochloride; F344: Fischer 344; HCE: hexachloroethane; IBT: isobutene;
dilatation of the left atrium, bradycardia, a low cardiac out-
put, and hypercoagulability generally summarize the causes
of formation of left atrial thrombus in human cases; sys-
temic diseases, such as malignant tumors, amyloidosis, and
nephritic syndrome, have also contributed to development of
intracardiac thrombus (Aoyagi et al., 2002). Atrial thrombo-
sis sometimes occurs as a side effect after therapy involv-
ing central venous catheterization, heart valve replacement,
or maintenance hemodialysis (Platt et al., 1980; Rotellar
et al., 1996; Kingdon et al., 2001; Shapiro et al., 2002;
Douketis, 2003; Nishimura et al., 2003). Recently, a cyclo-
oxygenase-2(COX-2)-inhibitor, one of a category of nons-
teroidal anti-inflammatory drugs, was withdrawn from the
market because of a significant risk of cardiovascular throm-
botic complications, such as development of cardiac throm-
bus and myocardial infarction (Konstam and Weir, 1999;
Bing and Lomnicka, 2002; Schmidt et al., 2004). Inhibition
of COX-2 may lead to increased prothrombotic activity by
tipping the balance of prostacyclin/thromboxane in favor of
thromboxane, a prethrombotic eicosaid (Mukherjee et al.,
2001; Bing and Lomnicka, 2002). Thus, much attention has
been focused upon cardiac thrombosis as one of several side
effects induced by certain medical treatments in humans.
518 YOSHIZAWA ET AL.
Of approximately 85,000 chemicals registered for use
in the United States, with an additional 2,000 introduced
annually, complete toxicological-screening data are obtain-
able for only 7%; for 40%, no data are available (Bennett
and Davis, 2002). Although the NTP has compiled a large
database of incidences of lesions seen in chemical-treated
animals ?http://ntp-server.niehs.nih.gov/index?, atrial throm-
bosis in rodents was not reported to be increased (Elwell
and Mahler, 1999) until recently, when atrial thrombosis was
induced by exposure to bis(2-chloroethoxy)methane (CEM)
1998a, 2003a, 2003b; Ezov et al., 2002). We, therefore ex-
amined all of the NTP data for more than 500 chemicals and
identified 13 that appeared to induce atrial thrombosis. Sup-
plementary data presenting the names and structures of these
compounds, as well as their usages and effects in humans,
are provided online (Please see the end of the abstract for the
URL). In addition, the rates of spontaneous atrial thrombosis
TABLE 1.—Chemical-induced heart thrombosis in NTP studies.
probably related to
DiazoaminobenzeneD 16D 0, 12.5, 25, 50, 100,
M: 0/5, 0/5, 0/5, 2/5
(100)g, 5/5 (100), 5/5
M: 1/60 (0), 34/60 (79),
F: 2/60 (0), 23/59 (65),
M: 0/50, 0/50, 0/50, 6/50
F: 0/50, 0/50, 1/50 (100),
F: 1/50 (100), 3/50 (100),
6/48 (67), 9/44 (89)
Left atrium Methemoglobinemia
F 57W 0, 2500, 5000 ppm
Left atrium Systemic amyloidosisTR443
D 2Y 0, 40, 80, 160 mg/kg
Left atriumCardiovascular effecti
G 2Y 0, 35, 75, 150 mg/kgLeft atriumThrombocytosisj
I 14W 0, 31, 62.5, 125, 250,
F (250, 500 ppm): 0/1,
D 13W 0, 50, 100, 200, 400,
0, 500, 2000,
0, 80, 170, 330 ppm
M: 0/10, 0/10, 0/10, 0/10,
0/10, 3/10 (100)
M: 0/50, 1/50 (100), 3/50
(100), 6/50 (67)
M: 3/60 (67), 15/44 (73),
27/75 (74), 23/60 (100)
M: 0/50, 6/50 (67), 4/50
(100), 3/49 (100)
M: 5/50 (60), 4/35 (100),
18/65 (56), 18/50 (94)
M: 2/50 (0), 3/35 (67),
17/65 (76), 12/50 (83)
M: 1/50 (100), 2/3 (50),
5/5 (20), 10/50 (40)
M: 1/50 (0), 0/15, 5/50
Left atriumOn goingn
I 2YLeft atriumTR487
C.I. Acid Red 114
DW2YLeft atrium Multiple tumorsl
F 2Y 0, 500, 1000,
0, 70, 150, 300 ppm
Left atrium Hemolytic anemia
DW2Y Left atriumTR405
C.I. Direct Blue 15Ratsc
DW2Y0, 630, 1250,
0, 10000, 25000,
0, 10, 20 mg/kg
C.I. Pigment Red 23Ratsc
F 2YLeft atrium Hemolytic anemiaTR411
G 2Y Left atriumNone TR361
dD: dermal, F: feed, DW: drinking water, G: gavage, I: inhalation.
eD: days, W: weeks, Y: years.
fM: male, F: female.
gIncidence rate (%) of dead or sacrificed-moribund animals exhibiting thrombosis.
hMoribund rats sacrificed within 8 weeks after chemical exposure.
i13-week mouse study.
j14-week rat study.
kLahlou et al. (2004).
lNo presence of vascular tumors.
mTR No.: technical report number, TSRS No.: toxicity study report series number. All reports are available on the NTP web site ?http://ntp-server.niehs.nih.gov/index?.
nBattelle Columbus reports submitted to NTP-G004164-AD & G004164-AE, July and October, 2002.
these 90-day and 2-year toxicity studies. This article, one of
studies, focuses on the incidences and morphologic aspects
from the literature of other chemically induced atrial throm-
boses and potential mechanisms of induction. Such results
contribute to the identification of environmental substances
that may affect the induction of cardiac thrombosis.
METHODS USED IN THE NTP STUDIES
The NTP has customarily used 2-year and short-term, pre-
liminary rodent bioassays to assess the overall toxicity and
carcinogenicity of the tested chemical and to identify poten-
tial human carcinogens. We searched all of the NTP data for
more than 500 chemicals and identified 13 that appeared to
induce atrial thrombosis. Table 1 lists aspects of the study
design for each of these chemicals identified as possible in-
ducers of atrial thrombosis, such as the animal species and
the route, duration, and dose of the exposure. Using data
Vol. 33, No. 5, 2005
CHEMICAL-INDUCED ATRIAL THROMBOSIS519
from NTP rodent studies, we described the incidences of
thrombosis in overall and early sacrificed animals exposed
to these chemicals ?http://ntp-server.niehs.nih.gov/index?, in
addition to control animals. The standard bioassay included
male and female B6C3F1 mice and F344 rats, although other
strains were occasionally included, such as Swiss Webster
mice that were used in the study of oxazepam. Rodents were
typically exposed to a chemical at 6–8 weeks of age by a
route of exposure chosen for its relevance to the known or
suspected route(s) of human exposure. All procedures, care,
and treatment of animals were in accordance with the princi-
ples of humane treatment outlined by the National Institutes
of Health (Grossblatt, 1996).
Results from the testing of more than 500 chemicals in the
NTP studies led to the identification of 8 compounds, some
reported in the original investigations, that induced atrial
thrombosis in rodent models: bis(2-chloroethoxy)methane,
2-butoxyethanol, C.I. Acid Red 114, C.I. Direct Blue 15,
diazoaminobenzene, 3,3?-dimethoxybenzidine dihydrochlo-
ride, isobutene, and oxazepam. Moreover, our examination
induced this abnormality, although no description of the in-
creased incidence of thrombosis was noted in the reports of
the NTP studies: C.I. Pigment Red 23, diethanolamine, hex-
achloroethane, methyleugenol, and 4,4?-thiobis(6-t-butyl-m-
For pathological analysis, complete necropsies were per-
formed on all animals using standardized methodology. At
necropsy, all tissues, including masses and other macro-
buffered formalin for microscopical evaluation. After fix-
ation, the tissues were trimmed, dehydrated, cleared, and
paraffin-embedded. Five-µm-thick sections were mounted
and examined microscopically. According to the “Guides for
Toxicologic Pathology” (Ruben et al., 2000), we used the
following criterion for the diagnosis of atrial thrombosis:
the composition of fibrin, platelets, and mixed inflamma-
tory cells in the atrial lumen. All data, including those from
pathological examination, were obtained according to GLP
standards, underwent extensive pathology peer review by an
The function of endothelium-derived nitric oxide syn-
thase (eNOS) in early atrial thrombotic change was in-
vestigated. Paraffin-embedded heart sections from F344
rats, each of 10 controls and 10 or 13 animals exposed
dermally to 600 mg/kg CEM for 2, 3, 5, and 16 days
were analyzed immunohistochemically to determine the ef-
fects of treatment on the expression of eNOS ?http://dir.
derived from the mechanistic study of CEM-induced car-
formed using the avidin-biotin-peroxidase technique. Tissue
slides were deparaffinized in xylene and alcohols and placed
in a 3% hydrogen peroxide solution for 15 minutes. Anti-
gen retrieval was achieved by placing the slides in 1X citrate
buffer, pH 6.0 (Biocare Medical, Walnut Creek, CA), boiling
them in the decloaker, and cooling for 10 minutes. Rabbit
anti-NOS3 (eNOS) antibody (1:600, Santa Cruz Biotech-
nology, Santa Cruz, CA) was applied for 1 hour. The sec-
ondary antibody (1:800, Vector Labs Burlingame, CA) was
applied for 30 minutes at room temperature. The immuno-
histochemical localization was visualized by using liquid
DAB (3,3?-diaminobenzidine tetrahydrochloride) (DakoCy-
tomation, Carpinteria, CA), applied for 30 minutes at room
temperature according to instructions given in the Vectastain
zoo, MI). All incubations were performed in a humidifying
chamber. Slides were dehydrated and coverslipped with Per-
mount (Surgipath, Richmond, IL).
of the immunopositivity and the relative area of the sections
exhibiting staining in the endocardial cells in the left atrium
were graded by 2 pathologists using a scale ranging from
0 (−) to 2 (++) as follows: (0) = no specific immunohisto-
logical reaction visible in endocardial cells, (1) = up to 50%
of total area showing a weakly positive reaction, and (2) =
SPONTANEOUS OCCURRENCE OF ATRIAL THROMBOSIS
IN F344 RATS AND B6C3F1 MICE
Historical control data from the NTP reports of cardiac
thrombosis in both genders of B6C3F1 mice and F344 rats
are shown in Table 2. In 90-day studies of rats and mice,
no cardiac thrombosis occurred spontaneously. The rate of
occurrence of cardiac thrombosis in 2-year mouse studies,
however, was 20 of 2,798 males and 21 of 3,110 females (in-
cidence rates: 0.71% and 0.68%, respectively). In the 2-year
In the 2-year studies, the incidence rates of dead or sacrificed
moribund animals exhibiting thrombosis were 85% (17/20)
for male mice, 81% (17/21) for female mice, 88% (118/134)
for male rats, and 91% (40/44) for female rats. All of the car-
diac thrombosis in these data was atrial, of which the main
localization was left atrial. Although these historical control
data include results from all kinds of dosing routes, such as
feed, gavage, inhalation, dermal, drinking water, and vagi-
nal application, some difference(s) might, nonetheless, exist
among animals dosed by different routes (data not shown).
TABLE 2.—Incidences of cardiac thrombosis∗in control animals in NTP ro-
B6C3F1 MiceF344 Rats
Female Male Female
aRoutes included feed, gavage, inhalation, dermal, and drinking water.
bRoutes included feed, gavage, inhalation, dermal, drinking water, and vaginal.
cNumber of animals with lesion per number of animals examined.
fRate (%) of dead or sacrificed moribund animals per number of animals exhibiting
520YOSHIZAWA ET AL.
The spontaneous occurrence of atrial thrombosis, also
called auricular thrombosis, has been reported in several ani-
mice (Maita et al., 1988; Hagiwara et al., 1996; Elangbam
et al., 2002), cotton rats (Sorden and Watts, 1996), hamsters
(Liu and Tilley, 1980; Allen et al., 1985), monkeys (Wood
et al., 1978; Kessler and London, 1982; Allen et al., 1985),
dogs (Jubb and Kennedy, 1993; Ayers and Jones, 1978), and
cats (Jubb and Kennedy, 1993; Ayers and Jones, 1978; Liu
thrombosis reported was 65% in retired breeding BALB/c
female mice, probably related to abnormalities of blood co-
described in aged animals, including rats and mice, often as-
inflammation, mineralization, amyloid deposition, or degen-
erative myxoid lesions in heart valves (Sorden and Watts,
1996; Ruben et al., 2000; Elangbam et al., 2002). Our histor-
might be useful in the evaluation of possible chemical- and
in F344 rats and B6C3F1 mice, few explicit reports exist
incidence of spontaneous atrial thrombosis in F344 rats and
B6C3F1 mice, compared to that in other murine strains. The
relationship between the occurrence of atrial thrombosis and
other cardiac lesions, especially spontaneous cardiomyopa-
thy, is not clear, because animals with atrial thrombosis do
not always exhibit a severe degree of cardiomyopathy, and
animals with a severe degree of cardiomyopathy do not al-
ways develop atrial thrombosis (data not shown). Thus, our
ways be related to cardiomyopathy in F344 rats and B6C3F1
Genetic modifiers of the coagulation response have been
examined as risk factors in cardiac thrombosis using sev-
eral genetically modified mice: factor IX-overexpression
mice with an abnormal blood-coagulation system (Ameri
et al., 2003), tissue-type and/or urokinase plasminogen-
activator knockout mice with disturbances in the fibri-
nolytic/coagulation cascade (Christie et al., 1999), and
Atm/mmice showing a mutation in the antithrombin gene
(Dewerchin et al., 2003). Additional reports have presented
relationships between the occurrence of cardiac thrombo-
sis and gene-modified murine strains, including ja/ja (jaun-
diced) mice with a deficiency in erythroid β-sectrin (Kaysser
et al., 1997); β-tropomyosin-overexpression mice exhibit-
ing abnormal cardiomyocytic contraction and relaxation,
which culminates in altered blood flow and thrombus for-
mation (Muthuchamy et al., 1998); tumor necrosis factor-
α-overexpression mice with severely reduced cardiac out-
as a direct result of local expression of cytokines and up-
regulation of molecules, such as tissue factor (Bryant et al.,
1998); β2-adrenergic receptor-overexpression mice exhibit-
ing severe myocardial hypertrophy and fibrosis (Du et al.,
2000); and the spontaneously hypertensive (SHR) rat, with a
hydrodynamic or rheologic problem in the lumen and struc-
tural changes in the vascular and cardiac walls (Nagaoka
a secondary change in dystrophic cardiac calcinosis (Everitt
et al., 1988); TS (Taconic Swiss), RF (Rockefeller Institute
strain), pregnant RF, and Swiss mice; and epinephrine- and
endotoxin-treated Holtzman rats (Ball et al., 1965; Clower,
1968; Clower and Douglas, 1968; Lockwood et al., 1969;
Renaud and Godu, 1969; Wicks et al., 1969; Clower and
1996) that develop fat-induced cardiovascular lesions, en-
docardial damage, or increased susceptibility of platelets to
thrombin-induced aggregation. Mural thrombosis might be
related to severe anemia induced by excessive fat in the diet,
since atrial thrombosis was prevented after therapeutic injec-
Dietary copper deficiency also induced atrial thrombosis in
Swiss Webster mice, acccompanied by cardiac enlargement
due to decreased coronary resistance and/or hemodynamic
overload related to severe anemia (Lynch and Klevay, 1994;
B6C3F1 mice and F344 rats has not been clearly related to
genetic or dietary factors.
CHEMICAL-INDUCED ATRIAL THROMBOSIS
Table 1 presents the results of our examination of the data
of these 13 compounds to determine the incidences and main
imals exhibiting cardiac thrombosis, the incidence was 20–
100% among the groups (Table 1). The incidence rate of
dead or sacrificed-moribund animals exhibiting thrombosis
was 78% (274/351 animals), similar to that of the sponta-
neous occurrence of thrombosis in control animals (Table 2).
in humans (Virmani et al., 2001; Lowe, 2004) and animals
(Jubb and Kennedy, 1993). The data that we uncovered sug-
gest that heart thrombosis could be the main cause of death
We formed 3 categories indicating possible relationships to
occurrence of multiple tumors.
RELATIONSHIP BETWEEN CHEMICAL-INDUCED ATRIAL
THROMBOSIS AND CARDIAC DAMAGE
Chronic heart failure in humans, known to confer upon
patients a greater risk of thromboembolism, is likely related
to numerous, diverse factors, such as vascular abnormalities,
output that promotes formation of fibrin-rich clots. In addi-
tion, defective endothelial function and significant levels of
bosis in humans can occur in association with cardiac dys-
function and abnormal blood flow (whirlpool) in the atrium
(Suetsugu et al., 1988; Bilge et al., 1999; De Lorenzo et al.,
2003). Autopsies and echocardiographic studies have indi-
cated near-50% incidences of thromboembolic events in pa-
tients with acute and chronic heart failures (Asinger et al.,
1981; De Lorenzo et al., 2003).
We were able to identify 3 chemicals from the NTP
database, which might induce atrial thrombosis secondary
to myocardial injury. We made this identification based upon
the progression and localization of this injury.
Vol. 33, No. 5, 2005
CHEMICAL-INDUCED ATRIAL THROMBOSIS521
bosis with myocardial degenerative change was noted in 3
of 10 male rats treated with 600 mg/kg CEM (Table 1).
In the time-course study of CEM-induced cardiac toxicity,
FIGURE 1.—Characterization of chemical-induced thrombosis in the left atrium. H&E. Asterisks show the thrombi. (a) Thrombosis in F344 rat exposed to
600 mg/kg bis(2-chloroethoxy)methane; animal from 13-week study died after 1 week of treatment. Thrombus occupies more than half of area of left atrium. Scale
bar = 2.5 mm. (b) Higher magnification of (a). Thrombus consists of fibrinous and inflammatory cellular debris. Subacute inflammation (arrow) and myocardial
degeneration (arrowheads) can be seen in left atrial wall. Scale bar = 100 µm. (c) Thrombosis in Swiss Webster mouse exposed to 5000 ppm OZP; animal from
(arrows) in all areas of heart, including left atrial wall. Scale bar = 2.0 mm. (d) Higher magnification of (c). Large thrombus with chondroid metaplasia (arrowhead)
2-butoxyethanol; moribund animal from 14-week study sacrificed 1 week after treatment began. Thrombus, located only at edge of left atrium (region termed the
atrial appendage), consists of fibrinous material mixed with inflammatory cells. Adherence of fibrinous materials to endocardial cells and hyperplasia of these cells
(arrows) can be seen. Scale bar = 100 µm. (f) Thrombosis in F344 rat exposed to 2500 ppm Blue 15; moribund animal from 2-year study sacrificed 93 weeks after
commencement of treatment. Large thrombus completely occupies the lumen of left atrium; fibrosis (arrows) and extensive mineralization (arrowhead) are apparent.
Scale bar = 100 µm.
severity of heart damage (NTP, 2003a). This heart lesion was
characterized by myocardial vacuolization, myocytic necro-
sis, mononuclear-cell infiltration, and fibrosis in all myocar-
dial areas, including the left atrium (Figure 1a, b) (Dunnick
et al., 2004a, 2004b). Damage in myocardial mitochon-
dria, constituting one of the earliest changes of myocardial
522 YOSHIZAWA ET AL.
damage, led to vacuolar formation (Dunnick et al., 2004a).
The mechanism of damage appears related to one of the
metabolites of CEM, thiodiglycoic acid, known to cause mi-
tochondrial damage in the myocardium.
In the mouse 2-year dermal study (NTP, 1999), higher in-
cidences of atrial thrombosis were noted in male and female
detected in 6 of 50 males and 4 of 50 females (incidence
rates: 12% and 8%, respectively) without other heart lesions;
however, atrial thrombosis was not attributed to chemical-
induced toxicity in the original report. In the previous 13-
week mouse studies, myocardial degeneration could be seen
in high-dose groups (NTP, 1992c; Melnick et al., 1994). The
possibility exists that DEL induced cardiac effects in the 2-
year mouse study, although myocardial damage was not de-
tected histopathologically. Atrial thrombosis may, therefore,
have been induced by DEL via cardiac functional changes.
In the 57-week study using Swiss Webster mice, the lower
survival of animals receiving OZP was attributable to an in-
crease in the extent and severity of amyloid deposits in many
organs. Atrial thrombosis and pulmonary lesions consistent
with chronic heart failure occurred at higher incidences and
with greater severity (Figure 1c, 1d) (NTP, 1993; Bucher
et al., 1994). The incidence of atrial thrombosis was 34/60
males (57%) and 23/59 females (40%) in the 2,500-ppm
groups and 35/60 males (58%) and 31/59 females (53%) in
the 5,000-ppm OZP-treated groups (Table 1). In CD mice,
systemic amyloidosis was seen, complicated with auricular
thrombosis (Maita et al., 1988). The Swiss Webster mouse
amyloidosis, which is related to chronic dermatitis (Gruys
reason that atrial thrombosis occurred only in the study us-
ing Swiss Webster mice might be related to the functional
damage induced by amyloidosis in the heart. Systemic amy-
loidosis in humans induced abnormal prothrombin time, fib-
rinogenopenia, increased fibrinolysis, abnormal coagulation
factors, and/or hemolytic anemia, accompanied by chronic
intravascular coagulation, probably due to vascular damage
produced by perivascular amyloid deposition (Bowie et al.,
1969; Santarone et al., 1999; Bick, 2001). A high prevalence
of atrial thrombosis was found in human patients with car-
diac amyloidosis, arising from atrial dysfunction ascribed to
the combination of amyloid infiltration of atrial walls and in-
creased atrial afterload caused by restrictive hemodynamics
(Santarone et al., 1999). Atrial amyloidosis has also induced
atrial fibrillation, followed by atrial stretch and endocardial
remodeling (Goette and Lendeckel, 2004).
Other Chemicals That Induced Atrial Thrombosis Related
to Myocardial Damage
Several chemicals have been reported to induce severe
myocardial damage, followed by atrial thrombosis. This
pathology has occurred at an incidence of up to 75% (left
treated intravenously with 4 mg/kg of doxorubicin (Fujihira
et al., 1993). An intraperitoneal injection of 100 mg/kg of
3-nitropropionic acid induced atrial thrombosis in several
strains of mice, but not B6C3F1 (Gabrielson et al., 2001).
Feeding of 0.1% hydrochlorothiazide (Lijinsky and Reuber,
1987) or more than 250 ppm quinacrine to F344 rats (Reuber
mentioned, the NTP conducted the toxicology and carcino-
genesis feeding studies of 2, using F344 rats and B6C3F1
mice: 3-nitropropionic acid (NTP, 1978) and hydrochloroth-
iazide (NTP, 1989b). In the NTP report of 3-nitropropionic
acid, an increased incidence of atrial thrombosis was not
noted, although this chemical induced myocardial damage
The reason for the differing results may be dependent on the
report on hydrochlorothiazide, the induction of atrial throm-
reasons were offered for the differences in the results of the
jinsky and Reuber, 1987) and the NTP study (NTP, 1989b).
In the Netherlands, the TNO conducted, in Wistar rats, a
29-month toxicity study of methyl bromide, which induced
atrial thrombosis accompanied by myocardial degeneration
(Reuzel et al., 1991). Causes of this atrial thrombosis were
suggested to be possibly related to mild sustained endocar-
dial damage and changes in the blood flow secondary to car-
diac muscular damage. Catecholamines induced myocardial
damage and intravascular aggregation of platelets (throm-
bus formation) in many species of animals, including rats
thrombosis occurred accompanied by myocardial damage,
RELATIONSHIP BETWEEN CHEMICAL-INDUCED ATRIAL
THROMBOSIS AND HEMATOLOGICAL CHANGES
phenomenon occurring with some association with throm-
bosis. Antineoplastic drugs, such as deoxycoformycin, pen-
tostatin, cisplatin, and mitomycin, have been associated in
humans with thrombotic thrombocytopenic purpura (TTP)
characterized by hemolysis and formation of microthrombi
in many organs, including heart (Bonner and Erslev, 1994;
Leach et al., 1999; Ezov et al., 2002; Dlott et al., 2004).
The most tenable hypothesis holds that TPP results from
the introduction into the circulation of one or more platelet-
aggregating substances due to immune-mediated or drug-
induced direct toxicity (Bonner and Erslev, 1994; Ezov et al.,
2002; Dlott et al., 2004). The direct toxicity of mitomycin
on endothelial function might play an important role in the
pathogenesis of TTP (Dlott et al., 2004). In human myeloma
probably due to sinus rhythm in the heart (Urbauer et al.,
2002; Jego et al., 2003). Retinoic acid induced intraventric-
ular thrombosis in human leukemia patients (Barbui et al.,
1998; Falanga et al., 2003). The mechanism has been in part
mediated by an increased expression of adhesion molecules
that facilitate adhesion of cells to vascular endothelium,
thereby promoting localized coagulation (Torromeo et al.,
Vol. 33, No. 5, 2005
CHEMICAL-INDUCED ATRIAL THROMBOSIS523
Several chemicals and drugs have induced methe-
moglobinemia, such as the antimalarials, chloroquine
and primaquine; local anesthetics (lignocaine, benzocaine,
and prilocaine); glyceryl trinitrate; sulphonamides; and
phenacetin (Coleman and Coleman, 1996; Hall et al., 1986).
The formation of methemoglobin induced by these chem-
icals, resulting in the production of blood clots, appar-
ently occurs predictably within a thrombus (Moody, 2003).
Drug-induced platelet antibodies have been demonstrated
to downregulate or enhance platelet function (Kekomaki,
2003). Sulfonamides induced immune thrombocytopenia,
decreased platelet production, or increased destruction of
platelets (Van den Bemt et al., 2004). Heparin-induced
with high levels of drug-dependent antibodies (Kekomaki,
From the NTP database, we were able to select 5 chem-
icals that might induce atrial thrombosis secondary to
or probably related to hematological changes, especially
In the 14-week rat inhalation study (NTP, 1998a), atrial
thrombosis without myocardial degenerative change was
Figure 1e). In the mechanistic study of BE-induced toxic-
ity, considerable evidence was reported of systemic dissem-
inated thrombosis in tissues including heart (atrium), nasal
cavity, incisor, liver, lung, femur, brain, eye, and/or tail (coc-
cygeal vertebrae), followed by infarction in some organs
et al., 2003, 2005; Redlich et al., 2004; Shabat et al., 2004;
Lewis et al., 2005). Hematological analysis indicated severe
hemolytic anemia and mild thrombocytosis. The thrombosis
in rats may represent a cycle of events in which an initial
low level of endothelial activation and/or dysfunction trig-
gered by hemolysis and hypoxia result in additional vascu-
lar problems, including enhanced erythrocytic adherence to
endothelial cells, overexpressions of vascular cell adhesion
molecule-1 (ICAM-1), poor blood flow, vascular occlusion,
thrombosis, infarction, and additional hypoxia (Koshkaryev
et al., 2003; Nyska et al., 2003; Potti et al., 2004; Redlich
et al., 2004; Shabat et al., 2004).
These histopathological changes in rats have provided
models of sickle cell anemia in humans (Ghanayem et al.,
2001; Ezov et al., 2002; Pathare et al., 2003). Metabolic acti-
vation of BE to form butoxyacetic acid (BAA) is a prerequi-
site for the development of hematotoxicity (Ghanayem et al.,
1987, 2001; Ghanayem and Sullivan, 1993). Human erythro-
cytes exposed to BAA have manifested changes similar to
those seen in rat erythrocytes, such as deformability, osmotic
fragility, and changes in sodium content, though human ery-
throcytes appear to be relatively resistant to the hemolytic
effects of BAA (Ghanayem, 1989; Ghanayem and Sullivan,
1993; Udden, 2002).
C.I. Pigment Red 23 (Red 23)
was not attributable to chemical-induced toxicity; however,
23-treated male group than in the control group (Table 1; in-
and the interim evaluations of the 2-year study (3 and 15
months), hemolytic anemia was noted at treatment levels
greater than 50,000 ppm. The presence of anemia in rats
and its absence in mice may be related to the difference in
life span of erythrocytes—50 to 65 days for rats versus 20
to 30 days for mice (NTP, 1992b). The short life span of
erythrocytes in mice enables them to replace damaged cells
faster than rats, thus maintaining these hematologic param-
eters within normal values. The mechanism of atrial throm-
bosis induced by Red 23 has not been clarified; however,
chemical-induced anemia may be attributable to the induc-
tion of atrial thrombosis.
dial changes in groups of males treated with 100 mg/kg or
more (Table 1; incidence: 5/5 males, 100 mg/kg; 5/5 males,
200 mg/kg). Clinical-pathology data indicated chemical-
induced methemoglobinemia, Heinz-body formation, and
hemolytic anemia. Methemoglobin is hemoglobin with iron
oxidized to the ferric (Fe+++) state from the normal, or re-
duced, ferrous (Fe++) state and rendered incapable of trans-
moglobin causes the production of blood clots (Moody,
2003). The higher incidence of atrial thrombosis might be
related to methemoglobinemia induced by DAB.
Our search for atrial thrombosis in the 2-year mouse study
revealed the higher incidence of this lesion in groups of fe-
males treated with 75 mg/kg or more of MEG. The incidence
showed 6/48 and 9/44 rats in the 75 and 150 mg/kg groups,
respectively (Table 1). In the NTP and other previous reports
in mice (Johnson et al., 2000; NTP, 2000; Abdo et al., 2001;
chemical-induced lesions, although the incidence was higher
in the 2-year study than that seen in control and background
data. Although hematological analysis was not conducted
in any mouse studies, evidence indicated the occurrence of
thrombocytosis, demonstrated by increased platelet counts
in the groups that received 100 mg/kg or greater in the 14-
week rat study (NTP, 2000; Abdo et al., 2001). Anemia was
marrow changes were noted in both rat and mouse studies;
therefore, anemia might have occurred in the 2-year mouse
studies. Disturbed blood flow may result from alterations in
the properties of erythrocytes, such as increased adherence
to the endothelium of the blood vessel wall (Koshkaryev
et al., 2003). Recent research showed that methyleugenol
elicited hypotension and bradycardia, effects that appeared
related to an active vascular relaxation (Lahlou et al., 2004).
The mechanism of the induction of atrial thrombosis has not
been elucidated; however, chemical-induced anemia, proba-
bly hemolytic, and cardiovascular effects, such as decreased
blood flow in the atrium, may contribute to the induction of
524 YOSHIZAWA ET AL.
In the NTP 2-year rat study (NTP, 1994), atrial throm-
bosis was not considered a chemical-induced effect; how-
ever, the incidence was increased in groups of males that re-
(Table 1; 12%, 8%, 6% incidence rates in 500, 1000,
2500 ppm-treated groups, respectively). Anemia and an in-
creased number of platelets induced by TBBC were noted in
the 2500 ppm-treated rat group and 1000 ppm-treated mouse
group. In the original report, alteration in platelets was ob-
posplenic or asplenic states, malignancies, acute blood loss,
and hyperadreno corticism. These associations appear to of-
in the induction of atrial thrombosis. While the mechanism
of induction has not been clarified, TBBC-induced anemia
(probably hemolytic) should be considered a possible cause
of atrial thrombosis.
RELATIONSHIP BETWEEN CHEMICAL-INDUCED ATRIAL
THROMBOSIS AND OCCURRENCE OF MULTIPLE TUMORS
In cancer of humans and animals, the development of
thrombosis involves a complex interaction between the tu-
mor cell, the patient, and the hemostatic system (Khato et al.,
1977; Tanabe et al., 1999; Schafer et al., 2003). Hyperfib-
rinogenemia contributes to the hypercoagulable state due to
a compensatory overproduction of clotting factors in cancer
patients (Khato et al., 1977; Tanabe et al., 1999). Tumor-
bearing mice given TNF exhibited intravascular clot forma-
tion with fibrin deposition in vivo. Activation of coagulation
of the matrix from TNF-stimulated human endothelial cells
was dependent on the presence of platelets, indicating their
important role in propagating reactions leading to formation
of fibrin in vitro (Tijburg et al., 1991; Jaimes et al., 2001).
coagulation (Ray, 2000; Philipp et al., 2003; Schafer et al.,
2003). Some kinds of malignant tumors, such as lymphoma,
stimulate megakaryocytopoiesis and platelet production dur-
by malignant tumors of an array of cytokines, such as IL-6,
a potent stimulator of platelet production (Ray, 2000).
In attempting to select chemicals from the NTP studies
that might be inducers of atrial thrombosis and probably in-
volved also in the occurrence of multiple tumors, we found
and describe 3 of them next.
C.I. Acid Red 114 (Red 114)
In the NTP 2-year rat study (NTP, 1991), atrial thrombosis
was noted among the chemical-induced lesions. The inci-
dence was higher, compared to controls, in groups of males
that received 150 ppm or more (Table 1; 28%, 36% in 150
bosis may have formed as a consequence of debilitation in
study and the interim evaluations (9 and 15 months) of the 2-
year rat studies, anemia—the poorly regenerative type–and
hypocellarity of bone marrow were noted, suggesting that
this chemical exerts a direct effect on hematopoietic cells
at high-dose levels. Chemical-induced multiple tumors were
liver; oral cavity; intestine; and lung. Some animals also de-
veloped mononuclear-cell leukemia. Although the mecha-
nism of the induction of atrial thrombosis has not been eluci-
dated, chemical-induced anemia and tumorigenesis may be
C.I. Direct Blue 15 (Blue 15)
In the NTP report (NTP, 1992a), atrial thrombosis was
noted among the chemical-induced lesions in rats, without
any description of the mechanism (Figure 1f). The incidence
groups, respectively). Blue 15-induced multiple tumors were
noted in skin; zymbal, clitoral, preputial, and adrenal glands;
liver; oral cavity; intestine; uterus; and brain. Some animals
developed mononuclear-cell leukemia. In the 3-, 9- and 15-
month rat studies, no induction of any cardiac lesions was
documented (Morgan et al., 1989; NTP, 1992a). The mech-
anism of the induction of atrial thrombosis by Blue 15 has
not been clarified, but we speculate that it might be related to
3,3?-Dimethoxybenzidine Dihydrochloride (DMOB)
In the NTP 2-year rat study (NTP, 1990), increased inci-
dences of atrial thrombosis in exposed males led to impaired
circulation and sludging of blood in the atrial chambers, as
well as increased morbidity. The incidence rates were 15/44
(34%), 27/75 (36%), and 23/60 rats (38%) in 80, 170, and
The compound induced many kinds of tumors in different
tissues (liver; intestine; zymbal, preputial, clitoral, and mam-
mary glands; oral cavity; skin; brain; uterus; mesothelium);
ical is one of the metabolites of Blue 15 (NTP, 1990, 1992a),
might be related to multiple tumorigenesis, as suggested for
Other Chemicals That Induced Atrial Thrombosis Related
to Vascular Tumors
Vascular tumors may sometimes be related to the patho-
genesis of thrombosis in other sites due to endothelial dam-
age, although the mechanisms are not clear (Miyamoto et al.,
1992). Multiple hemorrhagic and thrombotic episodes in-
volving vascular tumors can lead to thrombocytopenic and
hemorrhagic crises (Herman et al., 2002). The compound,
4-aminobiphenyl, induced left atrial thrombosis and heman-
giosarcoma in numerous organs in a mouse carcinogenesis
study (Schieferstein et al., 1985). Although the cause of the
induction may have involved a marked toxic effect on the
hematopoietic system, the relationship between the occur-
rence of atrial thrombosis and hemangiosarcoma should be
analyzed. In our investigation of 13 chemicals found in the
NTP database, no induction of vascular tumors in applicable
rodent studies involved chemical exposure.
Vol. 33, No. 5, 2005
CHEMICAL-INDUCED ATRIAL THROMBOSIS525
UNKNOWN MECHANISMS OF CHEMICAL-INDUCED
We could not speculate concerning the mechanism(s) of
atrial thrombosis induced by 2 chemicals obtained from the
changes or related lesions were induced.
An increased incidence of atrial thrombosis (incidence
in the NTP 2-year rat study (Table 1; NTP, 1989a); how-
1989a). No descriptions of hematological changes and car-
diovascular effects have been reported in rat studies (Weeks
et al., 1979; Gorzinski et al., 1985), similar to reports from
human cases (ATSDR, 1997).
In the NTP 2-year rat study (NTP, 1998b), the higher inci-
dence of atrial thrombosis in the 8000 ppm IBT-treated male
group was noted (12%, 6/50). The NTP decided, however,
that this marginal increase was not related to chemical expo-
was not conducted in this study.
Both of the aforementioned chemicals induced atrial
NTP historical background data. Both HCE and IBT should
be investigated further to distinguish between true and false
positivity and clarify the mechanism(s) of the induction of
RELATIONSHIP BETWEEN CHEMICALLY INDUCED ATRIAL
THROMBOSIS AND DAMAGE IN OTHER ORGANS
Severe renal disease sometimes results in secondary my-
ocardial fibrosis with the appearance of left atrial thrombosis
in more advanced cases (Glaister, 1986; Citak et al., 2000).
Researchers have speculated that lipoprotein in the human
nephritic syndrome may promote thrombosis (Stenvinkel
may be related to low levels of plasma antithrombin III and
albumin and high levels of fibrinogen and cholesterol (Citak
et al., 2000; Aoyagi et al., 2002). In our studies of 13 chem-
icals used to treat mice and rats, some compounds induced
renal lesions in the same groups in which the increased inci-
thrombosis, however, was not by itself related to chemical-
induced renal lesions (data not shown).
Spontaneous hippocampal neuronal necrosis that devel-
to vascular obstruction caused by atrial thrombosis or the
occurrence of leukemic cells and haemolytic anemia con-
comitant with mononuclear-cell leukemia, which commonly
no evidence could be discovered that atrial thrombosis was
related to the occurrence of any neural lesions or leukemia
induced by chemical exposure alone (data not shown).
induced left atrial thrombosis in F344 rats; dead animals
exhibited severe lung toxicity (Mitsumori et al., 1987). Al-
though this study was conducted by the NTP, we were un-
able to include this chemical in our investigation because
of the inability to locate the study findings at the given
web site ?http://ntp-server.niehs.nih.gov/index?. The mech-
anism of the induction of atrial thrombosis was not clari-
fied but, in the study report, was considered secondary to
alveolar damage or tissue hypoxia, because thrombus for-
mation was always restricted to the left atrium that drains
the pulmonary vein (Mitsumori et al., 1987). Large atrial
thrombosis, however, causes secondary passive pulmonary
lesions, such as congestion, sometimes with alveolar fibro-
plasias (Lewis, 1992). Thrombosis may not, therefore, occur
secondary to lung damage.
Certain infectious pathogens have long been suspected of
playing a role in the process leading to cardiac thromboem-
et al., 1985), bacterial endocarditis or myocarditis in mon-
keys infected by Staphylococcus or other bacteria (Kessler
and London, 1982; Wood et al., 1978), and chronic infec-
tion with Helicobactor pylori in mice resulting in increased
platelet embolization after damage to mesenteric arterioles
(Aguejouf et al., 2003). In our cases, no evidence was found
that atrial thrombosis was related to the occurrence of sys-
temic pathogenic infection (data not shown).
DIFFERENCES IN RESPONSES BY GENDER AND ANIMAL
TO CHEMICALLY INDUCED ATRIAL THROMBOSIS
Our research suggests differing responses to chemical-
induced atrial thrombosis of sex and strain of animals. The
reason for the gender differences in 2-BE-induced toxi-
city may be different rates of production of sufficiently
high levels of the hematotoxic metabolite, butoxyacetic acid
(Koshkaryev et al., 2003). With respect to the strain differ-
ence of the occurrence of myocardial damage induced by
CEM, the greater severity of CEM-induced heart toxicity
in rats than mice may have been due to their higher rates
of production of thiodiglycolic acid, a metabolite of CEM
(Dunnick et al., 2004b). No data were available, however,
from toxicokinetic studies of other chemicals, and differing
sensitivities by sexes and strains were not described in these
NTP studies. Future detailed investigations of the toxicoki-
netic characteristics of each chemical and its metabolite(s)
differences in chemically-induced atrial thrombosis.
FUNCTION OF ENDOTHELIUM-DERIVED NITRIC OXIDE
SYNTHASE (eNOS) IN EARLY CHANGE OF ATRIAL
murine thrombosis (Carter and Gavin, 1989; Triggle et al.,
marily from decreased synthesis of endothelium-derived NO
and/or an increase in the production of reactive oxygen
species, such as superoxide (Triggle et al., 2003; Davis et al.,
2004). Porcine atrial fibrillation causes a downregulation of
the production of atrial eNOS and NO and a comparative in-
crease in the expression of plasminogen activator inhibitor-1
in the left atrium during alterations comprising endocardial
526 YOSHIZAWA ET AL.
FIGURE 2.—Mean immunohistochemical scoring and morphological figures showing eNOS expression in left atrial endocardial cells in F344 rat exposed to
600 mg/kg bis(2-chloroethoxy)methane (CEM) for 2, 3, 5, and 16 days. (a) Mean eNOS scores.∗Significantly different (p < 0.05) from vehicle control group by
a 1 tailed Mann–Whitney test.∗∗p < 0.01. (b) Control animal from day-16 group. Nearly all endocardial cells are strongly positive for eNOS protein in cytoplasm
(arrows). Myocardial cells show weak cytoplasmic positivity. Scale bar = 50 um. (c) Animals treated with CEM for 16 days. No positive signals for eNOS can be
detected in the left atrial endocardium (arrows) or myocardial cells. Scale bar = 50 um.
remodeling; these collective changes were considered one of
the potential mechanisms for induction of left atrial throm-
bosis (Cai et al., 2002; Goette and Lendeckel, 2004). Nitric
and Lendeckel, 2004).
We analyzed the expression of eNOS in the left atrial en-
docardial cells in rats exposed to 600 mg/kg CEM for 2, 3,
5, and 16 days (Figure 2a,b,c). In the endocardial cells of
control rats, the mean scores were 1.7 to 2.2 (Figure 2a,b). In
contrast, the scores of animals treated with CEM for 2 and
16 days decreased with statistical significance and were 1.4
and 0.7, respectively (Figure 2a,c). Histological evaluations
indicating the significance of damage at these time points
have been described (Dunnick et al., 2004a). In addition,
atrial myocardial cells exhibited weak positivity for eNOS in
ported previously (Balligand and Cannon, 1997). Treatment
with CEM for 13 weeks induced left atrial thrombosis in rats
(Table 1). Our data and review of the literature suggest that
be an important and critical factor involved in early changes
leading to thrombus formation. Additional investigations are
necessary to clarify the relationship(s) between eNOS ex-
pression and the induction of atrial thrombosis by the other
chemicals that we have listed in this paper.
WHY SPONTANEOUSLY OCCURRING AND
CHEMICAL-INDUCED ATRIAL THROMBOSIS DEVELOPS
MAINLY ON THE LEFT SIDE
Data from the NTP rodent studies show that both sponta-
occurred mainly on the left side (Tables 1 and 2). Several re-
searchers have addressed the reasons for such localization in
humans and animals (Ayers and Jones, 1978; Lewis, 1992;
Al-Saady et al., 1999; Bilge et al., 1999; Elwell and Mahler,
1999; Ruben et al., 2000; Cai et al., 2002). In humans, left
atrial thrombosis has usually been considered the source of
embolic events in acute infarction (Bilge et al., 1999), al-
atrial thrombosis occurred as a terminal event in atrial fibril-
stasis of blood in the atrium or its auricular appendage (Jubb
and Kennedy, 1970). The incidence of thrombosis in atrial
fibrillation implies a role for an atrial hemodynamic factor;
atrial thrombosis has been associated with atrial fibrillation-
induced structural changes in the atrium, such as decreased
Vol. 33, No. 5, 2005
CHEMICAL-INDUCED ATRIAL THROMBOSIS527
contraction and dilatation of the atrial appendage (Bankl
et al., 1995; Al-Saady et al., 1999; Goette and Lendeckel,
atrial fibrillation were seen within the left atrial appendage
in human patients (Al-Saady et al., 1999).
Atrial fibrillation has been manifested in baseline electro-
cardiographs as cardiac rhythm showing irregular undula-
tions of varying amplitude, contour, and spacing (Aronow,
2002). Also a common complication of cardiac opera-
tions leading to increased risk for thromboembolism, it is
attributable to age-related structural changes in the human
atrium, such as dilatation, muscular atrophy, decreased con-
duction throughout tissue, and fibrosis (Hogue and Hyder,
2000). The left atrial appendage, a long, tubular, hooked
usually crenellated, with a narrow junction with the venous
FIGURE 3.—Mechanisms that may have contributed to chemical-induced cardiac thrombosis in the left atrium in the NTP rodent studies.
component of the atrium (Al-Saady et al., 1999); and closely
related in its superior aspect to the pulmonary artery and in-
feromedially to the free wall of the left ventricle (Al-Saady
et al., 1999; Nishimura et al., 2003). A possible reason for
the onset of fibrillation may be stagnation of blood in the left
atrial appendage (Suetsugu et al., 1988), which is a muscular
chamber acting as a contractile pump with a characteristic
pattern of contraction (Bilge et al., 1999). In rodents, this ap-
pendage appears to have a location similar to that in humans;
cardiac hypertrophy occurs therein at an incidence of 77%
in F344 rats aged 9 to 27 months (Boluyt et al., 1999). Lo-
cated that the left atrial appendage significantly contributes
to left ventricular filling and plays a pivotal role in main-
taining normal cardiac status, especially in states of cardiac
528 YOSHIZAWA ET AL.
disease (Kruse et al., 2001). The reason for the predilection
for the appendage in atrial thrombosis may involve not only
its distinctive anatomy, with the inner surface marked by
muscular ridges, but also abnormalities in blood-flow pat-
terns (Aronow, 1991; Bilge et al., 1999). Collectively, all of
this information indicates that spontaneously occurring and
chemical-induced murine atrial thromboses occur chiefly in
the left side because of distinctive anatomical and hemody-
namic characteristics of that region.
MECHANISMS OF CHEMICAL INDUCTION OF ATRIAL
THROMBOSIS AND POSSIBLE HUMAN RISK
Mechanisms of putative pathogenesis of thrombosis
have been indicated by several specific effects caused
by thrombosis-producing compounds: endothelial damage
(homocysteine, endotoxin, sodium acetriozate); alterations
in pathophysiologic circulatory dynamics (ergotamine,
pitressin, oral contraceptives, acetylcholine, autonomic
blockers); changes in platelets (serotonin, progesterone,
testosterone, somatotropic hormone, vincristine, congo
red, ristocein, thrombin, epinephrine, adenosin diphos-
phate, Evans blue); and transformations in clotting factors
(epinephrine, guanethidine, debrisoquin, thyramine, lactic
acid, long-chain fatty acids, catecholamines, ACTH, thy-
moleptics, nictotine, oral contraceptives, mercuric chloride,
corticosteroids, aminocaproic acid, aprotine) (Ramos et al.,
hypercoagulability—even though indirect, or secondary, ef-
to cardiac thrombi. The resulting impaired atrial mechanical
activity, occurring as atrial fibrillation and congestive heart
failure, might cause stasis of blood within the left atrium,
contributing to left atrial thrombosis (Figure 3).
There are reports in the literature of human left atrial
Goette and Lendeckel, 2004). No reports are available con-
one of the 13 compounds that we have reported to be associ-
ated with atrial thrombosis in the NTP studies. Some COX-
2-inhibitors, such as Vioxx, pose a significant risk of cardio-
Bing and Lomnicka, 2002; Schmidt et al., 2004). Preclinical
studies of these compounds did not reveal any potential risk
inability to detect potential risk of thrombotic development
in laboratory animals exposed to COX-2 inhibitors. Potential
factors may include suitability of the animal model, doses
selected for the testing, and different mechanisms leading to
development of thrombosis.
Future detailed investigations of hematological and elec-
trocardiological functions following exposure must be con-
real inducers of cardiac toxicities. To concentrate on molec-
ular functioning in such investigations could enhance un-
derstanding of the pathogenesis of chemical-induced atrial
thrombosis, since the progression and risks to humans of
this toxicity remain to be completely elucidated. Additional
research must be completed to analyze the precise mecha-
nism(s) of induction and provide understanding of potential
extrapolations from rodents to humans of chemical-induced
We gratefully acknowledge Dr. Micheal P. Jokinen of
Pathology Associates–A Charles River Company and Drs.
June Dunnick and Robert R. Maronpot of the NIEHS for
critical review of the manuscript. The authors declare that
they have no competing financial interests.
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