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
- NOTE: Click on images for larger views. Use
browser's "Back" button to return to this page.
Return to WSC Case Menu
- Case I 99133002 (AFIP 2676141)
- Signalment: 6-week-old, male, Sprague Dawley rat,
- 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
- 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,
- Conference Note: The differential diagnosis for nephrosis
generally includes: heavy metals, nephrotoxic xenobiotics (aminoglycosides
in particular), ischemia and smaller hydrocarbons (such as chloroform
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
- 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,
- 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,
- 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:
- Contributor's Diagnosis and Comments: Liver, oval
cell hyperplasia, moderate to marked with hepatocellular atrophy,
- 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,
- 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,
- 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:
- Laboratory Results:
Tests: Urinalysis, dip stick and slide (postmortem sample)
about 250 ery/uL
+++, about 50 mg/dL
- 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
- Case IV - 01711 (AFIP 2677996)
- Signalment: Full mouth (adult), Romney, female sheep
- 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
- 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,
- 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,
- 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.
- Return to WSC Case Menu