Rodent Liver Tumours and Human Health Risk Assessment

Rhian Cope

(A)Low-power photomicrograph of an AHF in a control mouse, which is recognizable as dysplastic under higher power (magnification, 63; bar = 100 µm).

(B)Higher magnification of AHF in (A) illustrating dysplasia including nuclear enlargement, increased nuclear/cytoplasmic ratio, nuclear hyperchromasia, variation in nuclear size and shape, irregular nuclear borders, and nucleoli that are increased in size and number with irregular borders (magnification, 250; bar = 100 µm).

Progression to Neoplasia

(C) LFCA in a liver from a mouse treated with 1 g/L DCA; note irregular border and lack of compression at edge (magnification, 63; bar = 100 µm).

(D) Higher magnification of LFCA in (C) illustrating a focus of dysplastic cells within the LFCA (magnification, 400; bar = 100 µm).

Progression to Neoplasia

(E) Edge of a large AD from a mouse treated with 3.5 g/L DCA, demonstrating compression of adjacent parenchyma and "pushing" border of lesion (magnification, 63; bar = 100 µm).

(F) Higher magnification of AD in (E) illustrating dysplastic cells (magnification, 400; bar = 100 µm).

Progression to Neoplasia

Carcinoma

Progression to Neoplasia

Foci of Hepatocellular Alteration: “Pre-neoplastic” change

Society of Toxicologic Pathology Classifications:• Foci of hepatocellular alteration:

• Basophilic cell foci, tigroid type and homogenous type – increased RER and decreased cell glycogen;

• Eosinophilic (acidophilic) cell foci – deficient in glucose-6-phosphatase; ground glass appearance;

• Clear cell foci – large unstained cytoplasm with no vacuoles;

• Amphiphilic cell foci – intensely eosinophilic cytoplasm;

• Mixed cell foci.

Basophilic FHA

Eosinophilic FHA

Clear Cell FHA

Mixed FHA

Foci of Hepatocellular Alteration: “Pre-neoplastic” change

Occur spontaneously with age in rats; also occasionally in dogs & non-human primates;

Type and number of spontaneous foci vary with strain;

Have the characteristics of initiated ± promoted cells;

Number increase with exposure to genotoxic carcinogens;

Represent an “adaptation” of the hepatocytes to a hostile environment i.e. maladaptive response;

Foci of Hepatocellular Alteration: “Pre-neoplastic” change

• Often express placental glutathione S-transferase (GST-P) and are UDP-glucuronosyltransferase negative in rats. Variable expression patterns found in mouse foci;

• Elevated replicative DNA synthesis;• Altered expression of various growth factors;• Over responsive to mitogens;• Inherent defects in growth control (i.e. becoming autonomous in

terms of growth);• Genomic instability;• Aberrant methylation of p16 TSG;• Mutations of ß-catenin; • Decreased apoptosis;• Clonal origin demonstrable in vitro

GST-P Positive FHA

Foci of Hepatocellular Alteration: “Pre-neoplastic” change

Relevance to humans:

• Similar pre-neoplastic foci occur in humans exposed to hepatic carcinogens (both viral and chemical);

• Also occur with non-genotoxic hepatocarcinogens i.e. anabolic steroids;

• Potentially relevant to humans depending on the mechanism!

Foci of Hepatocellular Alteration: “Pre-neoplastic” change

Reversibility:

• In the case of chemically stimulated FHA’s, a high proportion will partially or near-completely regress when the stimulus is removed;

• Meet the criteria for “initiation + promotion”;

• Initiation is irreversible, but initiation is not phenotypically detectable;

FHA Versus Focal Nodular Regenerative Hyperplasia and Nodular Regenerative Hyperplasia

Key differences:

• Cells phenotypically normal;

• Circumscribed i.e. not invading surrounding normal tissue;

• May be divided into pseudolobules by fibrous tissue (focal nodular regenerative hyperplasia);

• Not pre-neoplastic.

BUT: Can be very difficult to distinguish from AHF!

Foci of Pancreatic Tissue

Metaplasia NOT neoplasia;

Islands of seemingly “normal” exocrine pancreatic tissue within the liver;

Induced by Arochlor1254;

Focal hepatocyte adenoma

Adenoma Acinar Type(An adenoma is a benign tumor (-oma) of glandular origin)

Adenoma Trabecular Type(An adenoma is a benign tumor (-oma) of glandular origin)

Adenoma – Human Vs Rodent

Rodent• Clearly distinguishable from regenerative hyperplasia;• Usually larger than one lobule;• Compress the surrounding tissue;• Loss of normal lobular architecture but portal triads may be present;• Usually multifocal;• Not encapsulated with fibrous tissue;

• Humans• Difficult to differentiate from regenerative hyperplasia;• Usually solitary;• Usually encapsulated.

Carcinoma

Carcinoma: Carcinoma refers to an invasive malignant tumor consisting of transformed epithelial cells. Alternatively, it refers to a malignant tumor composed of transformed cells of unknown histogenesis, but which possess specific molecular or histological characteristics that are associated with epithelial cells, such as the production of cytokeratins or intercellular bridges.

Carcinoma Trabecular Type (Malignant)

Carcinoma Acinar Type (Malignant)

What is this??

Carcinoma Clear Cell Type (Malignant)

Carcinoma Scirrhous Type (Malignant)

Carcinoma Poorly Differentiated (Malignant)

What is so important about this?

Carcinoma – Human Vs Rodent

• Humans• Mixed cell tumors are relatively common;• Concurrent cirrhosis is common;• Usually associated with chronic hepatitis;• Rarely spontaneous – usually a history of viral exposure

and/or aflatoxin exposure and/or alcohol exposure.

Carcinoma – Human Vs Rodent

Rodent• Classically metastasize to lung (why?);• Derive from oval cells (pluripotent stem cells) in the periportal

area;• Mixed cell tumors (i.e. hepatocyte plus bile duct cell

carcinomas) do not occur• Usually do not involve concurrent cirrhosis or chronic hepatitis;• “Spontaneous” in older animals (also in hamsters and beagle

dogs);• “Spontaneous” tumors are common, particularly in some

strains.

So what sort of tumor is this?

ILSI/HESI MOA Framework

Is the weight of evidence sufficient to establish the MOA in animals?• Genotoxic?

• Potentially relevant to humans, particularly if tumors at multiple sites;

• Nongenotoxic?• Relevance to humans is highly dependent on the

mechanism!

ILSI/HESI MOA Framework

Are the key events in the animal MOA plausible in humans?

• Genotoxic• Do the mutations occur in human cells in vitro and in vivo?• Do the same spectrum of mutations occur?• Is the genotoxic progression similar?

• Histopathology• Is the same histopathological life history present in rodents

and humans?

ILSI/HESI MOA Framework

Are the key events in the animal MOA plausible in humans?

• Nongenotoxic?• Relevance is HIGHLY dependent on the mechanism;

• Do the hyperplastic effect + antiapoptotic effect occur in humans?

• If a receptor-mediated pathway is involved, is this pathway present in humans and of similar pathophysiological relevance?

• Is there a clear dose threshold and what is its relationship to human exposure?

ILSI/HESI MOA Framework

Taking into account kinetic and dynamic factors, are the key events in the animal MOA plausible in humans?

• TK is sufficiently similar to result in relevant concentrations at the site of action?

• Promutagens activated to the same extent in humans (i.e. TD issues)? (TD encompasses all mechanisms through which the concentration/amount at the site of action elicits the toxic effect);

• If redox damage is critical, does similar metabolism/events occur in humans?

• Do the tumors occur in a non-rodent species?

• Observation of tumours under different circumstances lends support to the significance of the findings for animal carcinogenicity. Significance is generally increased by the observation of more of the following factors:

•Uncommon tumour types; •Tumours at multiple sites; •Tumours by more than one route of administration; •Tumours in multiple species, strains, or both sexes; •Progression of lesions from preneoplastic to benign to malignant;

•Reduced latency of neoplastic lesions; •Metastases (malignancy, severity of histopath); •Unusual magnitude of tumour response; •Proportion of malignant tumours; •Dose-related increases;•Tumor promulgation following the cessation of exposure.

Relevance Depends on MOA