Signalment:  

Mature, male, Wistar-Han, rat (Rattus norvegicus).This rat was part of an exploratory toxicity study; it was approximately eight weeks old at study initiation. The rat was administered a once weekly intravenous bolus of doxorubicin (3 mg/kg) for 6 weeks and was found dead on study day 52, a few hours before the scheduled euthanasia/necropsy.

Gross Description:  

Macroscopic findings in these animals included small testes and epididymides which correlated with marked to severe degeneration of the seminiferous tubules and epididymal oligospermia histologically. Some animals were also observed to have a gelatinous edematous pancreas.

Histopathologic Description:

Heart: Within the heart there is severe cardiomyocyte degeneration in the left atrium with marked vacuolation, and lesser myofiber disorganization, fragmentation and hypereosinophilia. Rarely, myofiber necrosis is present, with necrotic myocytes exhibiting apoptotic or karyorrhectic nuclei. Vessels within the left atrium have plump reactive endothelium and contain marginating neutrophils. A 4-6 mm diameter laminated fibrin thrombus distends the left atrium and is multifocally adherent to the endocardium. The margin of the thrombus contains numerous degenerate and viable neutrophils, and abundant apoptotic debris. Depending on the section, rare to large colonies of 1-2 μm bacterial coccobacilli are noted within the thrombus, extracellularly or phagocytosed by leukocytes. The left atrial endocardial endothelium is plump and reactive, multifocally eroded, and the endocardium is expanded by transmigrating neutrophils. Additionally, there is minimal to mild cardiac myofiber degeneration with myofiber vacuolation and myofibrillar disorganization within the septum, left ventricle and right atrium.

Additional histologic lesions in these rats referable to doxorubicin administration are present in the kidneys, testes, epididymides and lungs. Kidney lesions included tubular degeneration/ regeneration, glomerular atrophy and vacuolation and hyperplasia/ hypertrophy of Bowmans capsule. Testicular lesions consisted of germ cell loss, with only Sertoli cells remaining within seminiferous tubules in severe cases. A secondary oligospermia was present within the epididymides. Within the lung there was degeneration, with prominent vacuolation, of the muscular media in large arteries, sometimes accompanied by inflammation and fibrinoid necrosis.

Morphologic Diagnosis:  

1. Heart: Multifocal cardiac myocyte degeneration with prominent vacuolation, and rare necrosis.
2. Heart, left atrium: Atrial thrombosis and neutrophilic endocarditis, with intralesional bacteria.

Condition:  

Doxorubricin toxicity

Contributor Comment:  

Doxorubicin (adriamycin) is an anti-cancer drug with a very wide antitumor spectrum, and efficacy against both solid tumors and haematological malignancies.(2,5) However, use of the drug is limited by the frequent occurrence of dose-dependent cardiotoxicity which produces cardiomyopathy and secondary congestive heart failure.(2) Acute doxorubicin toxicity in patients includes gastrointestinal complaints, cardiac arrhythmias, phlebitis and tissue necrosis from paravasal leakage, and hypersensitivity reactions.(5) Delayed toxicity includes myelosuppression, alopecia and cardiomyopathy, with chronic cardiotoxicity manifesting as congestive heart failure. (5) Doxorubicin has been used for nearly three decades as a chemotherapeutic, but only recently have some of the cytotoxic mechanisms of the drug been elucidated.(5) These include free radical formation, membrane lipid peroxidation, iron-dependent oxidative damage to macromolecules, direct DNA damage or interference with DNA repair, mitochondrial damage and induction of immune reactions involving antigen-presenting cells in the heart.(1,2) Free radical formation and redox cycling associated with doxorubicin treatment cause the generation of reactive oxygen species such as superoxide anion, hydrogen peroxide and hydroxyl radical.(2) Tissues with a less developed antioxidant defense mechanism, like the heart, are highly susceptible to anthracycline-induced oxygen radicals.(2)

The doxorubicin-induced lesions in this study increased in incidence and/or severity with increasing duration of dosing. Both the cumulative effect and the morphologic characteristics of the lesions were consistent with findings reported in the literature. The myocardial degeneration induced in the ventricles and septum in this study in rats was representative of that reported in mice in the literature; however, atrial lesions were much more severe. A similar pattern was described previously in mice treated with doxorubicin,(3) although the bulk of the literature describing heart lesions caused by doxorubicin is based on examination of transverse tissue sections taken through the midventricular and septal areas, which do not include evaluation of the atria. In mice, vacuolation and degeneration of atrial myocytes are shown with electron microscopy to be dilation of the sarcoplasmic reticulum and increased numbers of normal and/or degenerate mitochondria.(3) Atrial interstitial inflammatory cell infiltrates within the myocardium and endocardium are also reported.(3) Additionally, mice treated with doxorubicin show an incidence of atrial thrombosis approaching 75%.(3) The proposed cause of the atrial thrombosis in the mice was endothelial inflammation accompanied by abnormal blood flow secondary to the myocardial damage.(3)

JPC Diagnosis:  

Heart, atria: Cardiomyocyte vacuolar degeneration, necrosis and loss, diffuse, mild to severe with left atrial thrombosis and rare regeneration.

Conference Comment:  

The contributor provides an excellent review of doxorubicin toxicity. The moderator highlighted the point that the contributor makes above; that is, although doxorubicin toxicity is traditionally associated with ventricular, rather than atrial, cardiomyocyte vacuolization and degeneration, this is related to tissue sample sectioning rather than to a change in pathologic mechanism of doxorubicin. The atrial lesions in previous studies were likely not noted earlier because the atria were not sectioned for histopathologic examination. This underscores the complexity of toxicologic pathology and the importance of being thorough in toxicity studies. This case also represents a paradigm shift in how some participants evaluate and interpret the tissue changes, i.e. they initially speculated that doxorubicin could cause this type of lesion in the heart, but then discounted it as the etiology because the lesion is observed in the atrium rather than the ventricle. Lack of awareness that doxorubricin could induce lesions in the cardiac atria caused several participants to dismiss it as a possible etiology in favor of other toxic causes, thus illustrating the informational utility of this case.

Discussion then focused on the secondary effects of the histologic lesions in the atria. Residents concluded that the primary lesion is the myocardial change, which results in ineffective myocardial contraction, endothelial damage, release of prothrombotic substances and thrombosis. Three perturbations promoting thrombus formation, colloquially referred to as Virchows triad, are endothelial injury, altered normal blood flow (turbulence or stasis), and hypercoagulability.(4) Endothelial cell damage is not restricted to physical damage; any damage capable of disrupting the prothrombotic-antithrombotic balance favoring thrombosis is included in this category.(4)

Normal, non-activated endothelial cells have antithrombotic activity; activation or damage resulting from bacterial endotoxin, cytokines or changes in hemodynamic properties promote thrombus formation. The following chart summarizes the mechanisms for these actions:(4)

Antithrombotic Properties of Endothelial Cells		Prothrombotic Properties of Endothelial Cells
Antiplatelet effects	Covers thrombogenic subendothelial extracellular matrix (ECM) Produce PGI2 and nitric oxide inhibiting adhesion Produces adenosine diphosphatase to degrade ADP	Platelet effects	-�-� Endothelial injury exposes subendothelial ECM → platelet adherence via von Will
Anticoagulant Effects	-�-� Heparin-like molecules enhance thrombin inactivation via antithrombin III: 1. Thrombomodulin → binds thrombin → activates protein C → inactivates factors Va and VIIIa → inhibits clotting 2. Produces protein S and tissue factor pathway inhibitor → direct inhibition of factor VIIa (tissue factor) and factor Xa	Procoagulant effects	-�-� Cytokine (TNF or IL-1) or endotoxin → endothelial production of tissue factor → activation of the extrinsic clotting cascade -�-� Augment the catalytic function of activated factor IXa and Xa
Fibrinolytic effects	-�-� Produce tissue plasminogen activator → cleaves plasminogen to plasmin → leaves fibrin → thrombus dissolution	Antifibrinolytic effects	-�-� Secrete inhibitors of plasminogen activator → reduces fibrinolysis

Platelets play an equally important role in hemostasis. Once contact is made with extracellular matric (ECM) proteins, platelets have three functions:(4)

1. Adhere to collagen with glycoprotein Ib via vWF; additional glycoprotein receptors bind other ECM components.
2. Secretion of α- and dense-granule contents promoting thrombus formation. P-selectin-coated α-granules contain fibrinogen, fibronectin, platelet factor 4, PDGF, TGF-β, and factors V and VIII. Dense (δ) granules have ionized calcium, histamine, serotonin, epinephrine, ADP and ATP.
3. Platelets aggregate and change shape, forming the primary hemostatic plug.

The severity modifier in our morphologic diagnosis is intended to indicate that the lesion in the right atrium is mild, while the lesion in the left atrium is severe. There is likely some sectioning variability as participants did not see the bacteria described by the contributor.

References:

1. Arola OJ, Saraste A, Pulkki K, Kallajoki M, Parvinen M, Voipio-Pulkki LM. Acute doxorubicin cardiotoxicity involves cardiomyocyte apoptosis. Cancer Res. 2000;60:1789-1792.
2. Ayaz SA, Bhandari U, Pillai KK. Influence of DL α-lipoic acid and vitamin-E against doxorubicin-induced biochemical and histological vhanges in the cardiac tissue of rats. Indian J Pharmacol. 2005;37(5):294-299.
3. Fujihira S, Yamamoto T, Matsumoto M, et al. The high incidence of atrial thrombosis in mice given doxorubicin. Tox Pathol. 1993;21(4):362-368.
4. Mitchell RN. Hemodynamic disorders, thromboembolic disease and shock. In: Kumar V, Abbas AK, Fausto N, Aster JC eds. Robbins and Cotran Pathologic Basis of Disease. 8th ed. Philadelphia, PA: Elsevier Saunders; 2009:115-123.
5. Speth PAJ, van Hoesel Q, Haanen C. Clinical pharmacokinetics of doxorubicin. Clin Pharm. 1988;15:15-31.

Click the slide to view.

---

2-1. Heart

2-2. Heart, left atrium

---