AFIP Wednesday Slide Conference - No. 5
6 October 1999

Conference Moderator:
Dr. Keith Harris, Diplomate, ACVP
Product Safety Assessment, Searle
4901 Searle Parkway
Skokie, IL 60077
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Case I 99133002 (AFIP 2676141)
Signalment: 6-week-old, male, Sprague Dawley rat, (Rattus norvegicus)
History: These animals were part of a toxicologic study to evaluate the acute toxic effects of Gentamicin on kidneys of rats. The rats were given intraperitoneal injections of 100.0 mg/kg/day Gentamicin sulfate, then sacrificed and necropsied on day 9 post-inoculation.
Gross Pathology: The kidneys were mildly bilaterally enlarged, with surface and cortical pallor.
Laboratory Results: There was a fourfold elevation in mean serum creatinine on day 9. Urinary glucose excretion, as well as N-acetyl-b-glucosaminidase (NAG), m-glutamyl transpeptidase (GGT) and lactate dehydrogenase activities were all elevated on day 1 of dosing only.

Contributor's Diagnoses and Comments:
1. Kidney (proximal tubules), tubular degeneration and necrosis, acute to subacute, multifocal, patchy, severe with tubular regeneration, proteinosis, and hyaline droplets, phospholipidosis, rat.
2. Kidney, nephritis, interstitial, lymphoplasmacytic, multifocal, mild to moderate.

Microscopically, there is marked multifocal, patchy, primarily proximal tubular necrosis, degeneration and/or regeneration. Groups of proximal tubules have markedly attenuated epithelium, sloughed lightly eosinophilic, and granular luminal debris. Less affected tubules have varying degrees of flattening or swelling of the epithelium with increased granularity and vacuolation of the cytoplasm. Necrotic cells are frequently present adjacent to less affected cells in tubules of this type. Adjacent tubules have large, plump nuclei, multiple prominent nucleoli, basophilic to amphophilic cytoplasm, and frequent mitoses. In some tubules, variably sized hypereosinophilic globular droplets (consistent with hyaline droplets) are present in the cytoplasm and proteinaceous and cellular casts are frequently seen. Medullary tubular epithelia are unaffected, but occasionally contain protein casts and rare mineralization. The interstitium is multifocally infiltrated with mild to moderate numbers of lymphocytes and plasma cells, with fewer histiocytes and neutrophils.

Phospholipidosis is a storage disease characterized by the intracellular accumulation of excess phospholipids in tissues. Several cationic amphophilic drugs (CADs) have been reported as causes of phospholipidosis, including antibacterials, antifungals, antimalarials, antipsychotics, antidepressants, and cholesterol-lowering agents. Either a parent compound or its metabolite may be responsible for the accumulation of phospholipids. The CADs are chemically related by structure, all having a hydrophobic ring and a hydrophilic side chain with a positively charged (cationic) amine group. Phospholipidosis can also result from a genetic disorder (as is the case in Tay-Sachs disease and Sandhoff disease). Phospholipidosis has been described in humans and several animal species. Various tissues may be affected, with the pulmonary alveolar macrophages and pneumocytes being most frequently involved.
The cationic amphophilic drug, Gentamicin, is an aminoglycoside antibiotic, in which the primary target of toxicity is the proximal tubular epithelial cell of the kidney. The CADs are reabsorbed by endocytosis, bind to phospholipids in the brush border of the proximal tubule, and are stored in secondary lysosomes. Ultrastructurally, the hallmark of phospholipidosis is the presence of myelin figures in phagolysosomes, which appear as concentric multilaminated membranous whorls.
AFIP Diagnosis:
1. Kidney: Tubular degeneration, necrosis and regeneration (nephrosis), moderate, diffuse, with tubular proteinosis, Sprague-Dawley rat (Rattus norvegicus), rodent.
2. Kidney: Nephritis interstitial, subacute, multifocal, moderate.
Conference Note: The differential diagnosis for nephrosis generally includes: heavy metals, nephrotoxic xenobiotics (aminoglycosides in particular), ischemia and smaller hydrocarbons (such as chloroform and methoxyflurane).

Nephrosis has been defined as acute alteration of proximal tubular epithelium. Lesions progress from hyaline droplet degeneration with loss of brush border and dilated tubular lumina to necrosis. Different inciting agents may produce distinct patterns resulting from various mechanisms. Osmotic nephrosis, hyaline droplet nephrosis and lipidosis are common mechanisms.
Osmotic nephrosis results from exposure to osmotically active agents (such as mannitol, sucrose, and ethylene glycol), and is characterized by cytoplasmic vacuolation (hydropic change). Hyaline droplet nephrosis results from protein overload from glomerular disease or alpha-2-microglobulin nephropathy, and is characterized by hyalin, eosinophilic cytoplasmic droplets in tubular epithelium. Mild lipidosis has been reported in association with exposure to solvents, and is characterized by fat droplets within the cytoplasm of tubular epithelium.
Phospholipidosis occurs with administration of many xenobiotics, most commonly aminoglycosides, and is characterized by increased cytoplasmic lucency. Special procedures are required to diagnose phospholipidosis: toluidine blue staining of plastic-embedded sections demonstrates cytoplasmic bodies and electron microscopy demonstrates concentric multilaminated phospholipid membrane whorls referred to as "myelin figures". The intralysosomal myelin figures are thought to be remnants of membranes and organelles. Myelin figures are believed to form at an increased rate because of inhibition of protein synthesis Gentamicin. In this case, the nephrosis and the interstitial nephritis are considered to be two separate processes. Interstitial nephritis is very common in rats.
Contributor: Pfizer Inc, Central Research, Drug Safety Evaluation, Pathology, Eastern Point Road, Groton, CT 06340
1. Amdur MO, Doull J, Klaassen CD: Systemic pathology: toxic responses of the kidney. In: Casarett and Doull's Toxicology: The Basic Science of Poisons, McGraw Hill, 4th ed., pp. 371-2, 1991
2. Halliwell WH: Cationic amphiphilic drug-induced phospholipidosis. Toxicol Pathol, 25(1): 53-60, 1997
3. Haschek WM, Rousseaux CG: Urinary system. In: Handbook of Toxicologic Pathology, Academic Press, pp.357, 1991
4. Kodavanti UP, Mehendale HM: Cationic amphiphilic drugs and phospholipid storage disorder. Pharmacol Rev, 42(4): 327-54, 1990
Case II - 99R253A (AFIP 2676139)
Signalment: Fisher 344/N/male, 19 weeks old (#98), 112 g (Rattus norvegicus)
History: The rat was administered 5 once weekly injections intraperitoneally of 2'amino anthracene as part of 14 week study.
Gross Pathology: The liver was small (2.7 g: average normal 10 g) firm, and had rounded edges.
Laboratory Results:
 Test  Result Test Result
 Glucose  34 g/dl  Protein  3.4 mg/dl
 AST  361 u/l  Albumen  3.4 mg/dl
 ALT  251 u/L  Cholesterol  126 mg/dl
 SAP  564 u/L  BUN  66 mg/dl
 CPK  355 u/l  Creatinine  0.4 mg/dl
 Bilirubin  6.1 mg/dl    
Contributor's Diagnosis and Comments: Liver, oval cell hyperplasia, moderate to marked with hepatocellular atrophy, moderate.
This was the only one of more than 30 rats similarly treated that developed these microscopic changes. Rats in the same treatment group had mild hepatic nodular hyperplasia and some hepatocellular centrilobular hypertrophy. Oval cell hyperplasia has been reported as a preneoplastic lesion of the biliary system and has been associated with such hepatic carcinogens as Helicobacter sp. Oval cell proliferation is considered to be completely reversible.

AFIP Diagnosis: Liver: Hyperplasia, oval cell, diffuse, marked, with multifocal nodular hepatocellular hyperplasia, Fisher 344 rat, rodent.
Conference Note: Although there has been some controversy as to origin/lineage of oval cells and hepatocytes, it is generally accepted that oval cells may differentiate into hepatocytes ("mature" hepatocytes can replicate as well). At the margins of the hepatocyte nodules, some of the smaller, more amphophilic hepatocytes may represent atrophic cells or cells in the process of differentiation from oval cells to hepatocytes.

As hepatocyte hyperplasia and hypertrophy also were present in other rats in this study, it is possible the oval cell change and hyperplasia are both related to the toxicity of 2'amino anthracene. This possibility would be strengthened if the submitted sections are from a rat in the high dose group.
Although Helicobacter infection can produce similar morphologic changes in mice, we are not aware of reports of Helicobacter inducing this type of liver disease in rats. Helicobacter-associated lesions of this severity are usually seen in older mice, are not this uniform, usually have areas of mixed inflammatory cells, and have foci of necrosis.
This case was studied in consultation with Dr. Michael Elwell of Covance Laboratories.
Contributor: Louisiana State University, Department of Veterinary Pathology, Baton Rouge, LA 70803
1. Eustis SL, Boorman GA, Harada T, Popp JA: Liver. In: Pathology of the Fischer Rat, eds. Boorman GA, Eustis SL, Elwell MR, Montgomery CA, MacKenzie WF, pp. 71-94. Academic Press Inc., San Diego, CA, 1990
2. Goodman DG, Maronpot RR, Newberne PM, Popp JA, Squire RA: Proliferative and Selected Other Lesions of the Liver of Rats GI-5. In: Standard System of Nomenclature and Diagnostic Criteria Guides for Toxicologic Pathology, STP/ARP/AFIP, Washington, DC, 1994
3. Haschek WM, Rousseaux CG: Hepatobiliary system. In: Fundamentals of Toxicologic Pathology 2nd ed, pp 142-143 Academic Press. 1998
4. Hoover, KL: Oval Cell Hyperplasia, Liver, Mouse, Rat. In: Monographs on Pathology of Laboratory Animals, Digestive System, eds. Jones TC, Mohr U, Hunt RD, pp. 125-126. Springer-Verlag, Berlin, Germany 1985
Case III - 99-391 (AFIP 2679487)
Signalment: Adult, male, rhesus monkey (Macaca mulatta)
History: Oral administration of compound (via intubation); vomited twice over next 3 days; otherwise healthy and alert. On day 4 post administration animal was down in cage on all four limbs. Animal was given fluids subcutaneously. Animal died one hour later on day 4 post administration. Blood draws performed at 1+, 6+, 24+, 48+, 72+ hours post administration.
Gross Pathology: Liver: Diffuse centrilobular necrosis. Thoracic wall; pericardium; pancreas; thymus; diaphragm and subcutis: Multifocal ecchymoses.
Laboratory Results:
Tests: Urinalysis, dip stick and slide (postmortem sample)
Feature Result
 Color  dark yellow
 Specific Gravity  1.025
 Urobilinogen  Normal
 Occult Blood  about 250 ery/uL
 Ketones  negative
 Glucose  normal
 Protein  +++, about 50 mg/dL
 pH  5
Contributor's Diagnoses and Comments:
1. Liver, hepatocytes: Degeneration and necrosis, submassive, acute, diffuse, with severe congestion and hemorrhage, rhesus monkey, non-human primate.
2. Kidney: Tubular degeneration and necrosis, diffuse, moderate to severe, with multifocal cellular, granular and hemoglobin casts and tubular protein.

A male rhesus monkey received an oral treatment of a compound (7.0 mg/kg, prepared in Multisol, administered in volume of 2ml/kg) via oral intubation at 0910 on 21 April 99. He vomited several times on 22 and 23 April 99. Animal was observed down in his cage and hypothermic. He received subcutaneous fluids and heat therapy at 0800 on 25 April 99. At 1100 on 25 April 99 animal was observed on all four limbs, stiff and still. At 1215 on 25 April 99 animal was found dead in cage. Urinalysis from a sample collected during post mortem examination revealed elevated protein and the presence of occult blood.
The compound administered to this monkey is an 8-aminoquinoline derivative that is structurally similar to the anti-malarial drug, primaquine. This compound is under development as a prophylactic treatment for cyanide toxicity. Its mechanism of action involves the formation of methemoglobin, which preferentially binds cyanide and thereby diverts cyanide from metabolism by mitochondrial cytochrome oxidase preventing cell death.

The histologic lesions in the liver and kidney are consistent with acute toxicity. The presence of hemorrhage in multiple organs from this animal is compatible with a generalized coagulation disorder most likely secondary to severe liver dysfunction. Necrosis of the zona reticularis in the adrenal gland is also consistent with an acute toxic insult. The presence of hemoglobin in urine and renal tubules indicates intravascular hemolysis. The exact mechanism of hemolysis is unknown. The exact mechanism of toxic injury, whether an effect of the compound itself and/or a biotransformed metabolite, is uncertain.
AFIP Diagnosis:
1. Liver: Necrosis and hemorrhage, centrilobular, diffuse, with periportal hepatocellular vacuolar degeneration, rhesus monkey (Macaca mulatta), primate.
2. Kidney: Tubular epithelial degeneration and necrosis, diffuse, moderate, with granular casts.
Conference Notes: Conference participants generally agreed with the contributor's morphologic diagnoses and comments. The special terms used for describing hepatic necrosis were discussed. The following terms, descriptions and definitions were taken from Robbins Pathologic Basis of Disease. Ischemic coagulative necrosis is characterized by hepatocytes that are poorly stained, "mummified", and often have lysed nuclei. Councilman bodies are hepatocytes that are isolated, rounded up, shrunken, and intensely eosinophilic. They contain fragmented nuclei. This form of cell death is a result of apoptosis. Centrilobular hepatic necrosis affects hepatocytes around the terminal hepatic vein and is characteristic of ischemic injury and a number of toxic and drug reaction. Focal necrosis is limited to scattered hepatocytes within hepatic lobules. In interface hepatitis, hepatocyte necrosis is limited to the interface between the periportal parenchyma and the inflamed portal tracts.

More severe inflammatory injury may result in necrosis of contiguous hepatocytes and may span adjacent lobules in a portal to portal, portal to central, or central to central pattern termed bridging necrosis. Necrosis of entire lobules is submassive necrosis. Necrosis of most of the liver is massive necrosis.
Contributor: United States Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD
1. Crawford JM: The liver and biliary tract, In: Robbins Pathologic Basis of disease, 6th ed, Cotran RS, Kumar V, and Collins T, eds. pp. 847-847.
Saunders, Philadelphia, Pennsylvania, 1999
2. Marino MT, Peggins JO, Brown LD, Urquhart MR, Brewer TG: Pharmacokinetics and kinetic-dynamic modeling of an 8-aminoquinoline candidate anticyanide and antimalarial drug. Drug Met and Disp 22(3):358-366, 1994
Case IV - 01711 (AFIP 2677996)
Signalment: Full mouth (adult), Romney, female sheep (Ovis aires).
History: The subject is one of 1,250 mixed age ewes with suckling lambs from a mob of 50,000 which sickened and died following pasture contamination by tephra. Deaths commenced nine days following the October 1995 eruption of Mount Ruapehu and continued for the following ten days. Affected sheep exhibited depression, salivation, hyperpnea, inappetance, blindness, ataxia and death.
Gross Pathology: The carcass was in satisfactory general condition. The kidneys were pale and enlarged with gelatinous edema of the perirenal fibroadipose connective tissues. There was coarse, gray, gritty material within the rumen (typical of the volcanic tephra on the pasture and soil). Feces were mucoid.
Laboratory Results: The most consistent finding in all sheep tested was low serum calcium, 1.34-2.64 mmol/l (normal range 2.9-3.2). Otherwise the serum chemistry of typically affected sheep revealed severe azotemia and myonecrosis. Blood fluoride levels of the subject animal were 520mg/kg (toxic dose > 200mg/kg).

Contributor's Diagnosis and Comments: Kidney: Acute nephrosis.
Etiology: Acute fluoride toxicosis.
The mineral composition of volcanic ash and hence the potential toxic effects on livestock can differ considerably from eruption to eruption by the same mountain as well as from variations between mountains (Gregory and Neal, 1996). The Ruapehu series illustrates this point well, as in the following year no cases of fluorosis were identified in association with continued volcanic activity. While the death of the animal in this case can be explained principally on the basis of renal failure caused by fluorosis, it is likely that other factors such as hypocalcemia, secondary metabolic disorders and stress related diseases could also have contributed significantly to the demise of this and other animals in the flock (Shanks, 1997). Fluoride toxicosis has been reported as a cause of acute and chronic illness in livestock exposed to volcanic tephra. Fluorine is present as an absorbed outer layer on the tephra particles and is highly water soluble. The eruptions associated with the outbreak reported here coincided with a period of continuous rainfall and this, as a result of leaching, is thought to have reduced the threat of chronic fluorosis. However, localized outbreaks of acute disease were reported.
Serum fluoride was seldom elevated in the animals tested in the present outbreak, even when the rumen levels were excessive. Soluble fluoride is highly labile within the body, being rapidly absorbed and excreted. Urine and bone are therefore likely to be the most appropriate animal substances for analysis.
Histologically the outstanding feature is necrosis of the proximal convoluted tubules of the kidney, the changes being identical to those recorded in fluorosis associated with superphosphate toxicosis (O'Hara et al, 1982).
AFIP Diagnosis:
1. Kidney: Tubular epithelial degeneration and necrosis (nephrosis), diffuse, moderate, with tubular proteinosis and granular casts, Romney sheep (Ovis aires), ovine.
2. Kidney: Nephritis, tubulointerstitial, subacute, multifocal, mild.
Conference Note: While acute fluoride intoxication produces clinical signs and lesions of gastroenteritis and renal tubular necrosis, chronic fluoride intoxication may produce dental and/or skeletal lesions. Fluoride is metabolized via renal excretion and is preferentially deposited in bones and teeth. Sources of environmental fluoride include natural subsurface water where rock phosphate is plentiful, industrial effluent and dust from volcanic eruptions, as in this case. Dental lesions, which may include enamel hypoplasia, attrition of molars and crown loss, develop only if intoxication occurs when the teeth are in the developmental stages. The lesions associated with the skeleton may include kyphosis, formation of hyperostoses and osteodystrophy.
The differential diagnosis in this case includes heavy metal intoxication (arsenic, bismuth, cadmium, lead, mercury, thallium), ingestion of plants containing oxalates or tannins, mycotoxins, hypotension leading to renal ischemia, urinary outflow obstruction, aminoglycosides and chlorinated hydrocarbons.
Small numbers of crystals were seen within tubules in this case. Similar crystals are frequently seen within renal tubules of sheep and their presence is not considered significant.
Contributor: New Zealand Registry of Animal Pathology, Pathology Section, IVABS, Massey University, Palmerston North, New Zealand
1. Gregory NG, Neall VE: Toxicity hazards arising from volcanic activity. Surveillance. 23:14-16, 1996
2. O'Hara PJ, Fraser AJ, James M: Superphosphate poisoning of sheep: the role of fluoride. N. Z. vet. J. 30:199-201, 1982
3. Shanks D F: Clinical implications of volcanic eruptions on livestock - Case studies following the 1995 and 1996 eruptions of Mt. Ruapehu. Proceedings of the 27th Seminar of the Society of Sheep and Beef cattle Veterinarians. Veterinary Continuing Education, Massey University. 175:1-13, 1997
J Scot Estep, DVM
Captain, VC, USA
Registry of Veterinary Pathology*
Department of Veterinary Pathology
Armed Forces Institute of Pathology
(202)782-2615; DSN: 662-2615
* The American Veterinary Medical Association and the American College of Veterinary Pathologists are co-sponsors of the Registry of Veterinary Pathology. The C.L. Davis Foundation also provides substantial support for the Registry.
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