Gary A. Boorman

Use of Historical Control Data in Carcinogenicity Studies in Rodents

This paper considers the use of historical control data in the evaluation of tumor incidences from carcinogenicity studies in rodents. Although the most appropriate control group for interpretative purposes is always the concurrent control, there are instances in which the use of historical control information can aid an investigator in the overall evaluation of tumor incidence data. One example is rare tumors; another is a tumor that shows a marginally significant result relative to concurrent controls. However, before historical control data can be used in a formal testing framework, a number of important issues must first be considered. The nomenclature conventions and diagnostic criteria for each study should be identical to insure unambiguous identification of all relevant tumors in the historical control database. Criteria should be established that will aid in determining whether a particular study should be included in the database. This will assure a homogeneous set of studies upon which to base statistical comparisons. Since study-to-study variability in tumor rates may exceed what would be expected by chance alone, these sources of variability should be identified and controlled. Finally, statistical procedures should be employed that adjust for extra-binomial variability. This paper also summarizes tumor incidence data from untreated Fischer 344 rats and B6C3F1 mice in the National Toxicology Program (NTP) historical control database. All studies in the database are of two years duration, and all neoplasms occurring with a frequency of 0.5% or more are reported.

Arsenic Enhancement of Skin Neoplasia by Chronic Stimulation of Growth Factors

Although numerous epidemiological studies have shown that inorganic arsenicals cause skin cancers and hyperkeratoses in humans, there are currently no established mechanisms for their action or animal models. Previous studies in our laboratory using primary human keratinocyte cultures demonstrated that micromolar concentrations of inorganic arsenite increased cell proliferation via the production of keratinocyte-derived growth factors. As recent reports demonstrate that overexpression of keratinocyte-derived growth factors, such as transforming growth factor (TGF)-alpha, promote the formation of skin tumors, we hypothesized that similar events may be responsible for those associated with arsenic skin diseases. Thus, the influence of arsenic in humans with arsenic skin disease and on mouse skin tumor development in transgenic mice was studied. After low-dose application of tetradecanoyl phorbol acetate (TPA), a marked increase in the number of skin papillomas occurred in Tg.AC mice, which carry the v-Ha-ras oncogene, that received arsenic in the drinking water as compared with control drinking water, whereas no papillomas developed in arsenic-treated transgenic mice that did not receive TPA or arsenic/TPA-treated wild-type FVB/N mice. Consistent with earlier in vitro findings, increases in granulocyte/macrophage colony-stimulating factor (GM-CSF) and TGF-alpha mRNA transcripts were found in the epidermis at clinically normal sites within 10 weeks after arsenic treatment. Immunohistochemical staining localized TGF-alpha overexpression to the hair follicles. Injection of neutralizing antibodies to GM-CSF after TPA application reduced the number of papillomas in Tg.AC mice. Analysis of gene expression in samples of skin lesions obtained from humans chronically exposed to arsenic via their drinking water also showed similar alterations in growth factor expression. Although confirmation will be required in nontransgenic mice, these results suggest that arsenic enhances development of skin neoplasias via the chronic stimulation of keratinocyte-derived growth factors and may be a rare example of a chemical carcinogen that acts as a co-promoter.

Arsenic can mediate skin neoplasia by chronic stimulation of keratinocyte-derived growth factors

Although numerous epidemiological studies have shown that inorganic arsenicals are human skin carcinogens, there is currently no accepted mechanism for its action or an established animal model for its study. We observed increased mRNA transcripts and secretion of keratinocyte growth factors, including granulocyte macrophage-colony stimulating factor (GM-CSF) and transforming growth factor-alpha (TGF-alpha) and the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) in primary human epidermal keratinocytes cultured in the presence of low micromolar concentrations of sodium arsenite. Total cell numbers, as well as c-myc expression and incorporation of [3H]thymidine, both indicators of cell proliferation, were also elevated in keratinocyte cultures treated with sodium arsenite. As an in vivo model, the influence of arsenic on mouse skin tumor development was studied in transgenic TG.AC mice which carry the v-Ha-ras oncogene, and can serve as a genetically initiated model for skin carcinogenesis. Following low-dose application of 12-O-tetradecanoyl phorbol-13-acetate (TPA), a marked increase in the number of skin papillomas occurred in transgenic mice receiving arsenic in the drinking water as compared to control drinking water. Papillomas did not develop in arsenic-treated transgenic mice that had not received TPA or arsenic-treated wild-type FVB/N mice, suggesting that arsenic is neither a tumor initiator or promoter but rather an enhancer. Injection of anti-GM-CSF antibodies following application of TPA in transgenic mice reduced the number of papillomas. Consistent with that observed in human keratinocyte cultures, increases in GM-CSF and TGF-alpha mRNA transcripts were found within the epidermis of arsenic-treated mice when compared to controls within 6 weeks of treatment. These results suggest that arsenic enhances papilloma development via the chronic stimulation of keratinocyte-derived growth factors and represents the first example of a chemical carcinogen that acts in this manner. These studies suggest that in vitro studies with human keratinocyte cultures examined in conjunction with TG.AC transgenic mice can provide a useful model for examining the tumor enhancing properties of environmental chemicals.

Mouse population-guided resequencing reveals that variants in CD44 contribute to acetaminophen-induced liver injury in humans

Interindividual variability in response to chemicals and drugs is a common regulatory concern. It is assumed that xenobiotic-induced adverse reactions have a strong genetic basis, but many mechanism-based investigations have not been successful in identifying susceptible individuals. While recent advances in pharmacogenetics of adverse drug reactions show promise, the small size of the populations susceptible to important adverse events limits the utility of whole-genome association studies conducted entirely in humans. We present a strategy to identify genetic polymorphisms that may underlie susceptibility to adverse drug reactions. First, in a cohort of healthy adults who received the maximum recommended dose of acetaminophen (4 g/d x 7 d), we confirm that about one third of subjects develop elevations in serum alanine aminotransferase, indicative of liver injury. To identify the genetic basis for this susceptibility, a panel of 36 inbred mouse strains was used to model genetic diversity. Mice were treated with 300 mg/kg or a range of additional acetaminophen doses, and the extent of liver injury was quantified. We then employed whole-genome association analysis and targeted sequencing to determine that polymorphisms in Ly86, Cd44, Cd59a, and Capn8 correlate strongly with liver injury and demonstrated that dose-curves vary with background. Finally, we demonstrated that variation in the orthologous human gene, CD44, is associated with susceptibility to acetaminophen in two independent cohorts. Our results indicate a role for CD44 in modulation of susceptibility to acetaminophen hepatotoxicity. These studies demonstrate that a diverse mouse population can be used to understand and predict adverse toxicity in heterogeneous human populations through guided resequencing.

National Toxicology Program Nomenclature for Hepatoproliferative Lesions of Rats

Diagnostic criteria for hepatoproliferative lesions of Fischer 344 rats are presented to permit more complete categorization of the spectrum of lesions observed in two-year chemical carcinogenicity studies. A nomenclature recently adopted by the National Toxicology Program differs from previous classification schemes in that hepatocellular hyperplasia and hepatocellular adenoma are to be used for lesions which were previously combined under the diagnosis of neoplastic nodule. The term hyperplasia is reserved for proliferative lesions that are perceived to be secondary, nonneoplastic responses to degenerative changes in the liver. Foci of cellular alteration, hepatocellular adenoma, and hepatocellular carcinoma are believed to represent a spectrum of changes that comprise the natural history of neoplasia. This change in nomenclature was made subsequent to a peer review of representative hepatoproliferative lesions from two-year carcinogenicity studies. The revised nomenclature is consistent with traditional pathologic diagnoses for proliferative lesions in other epithelial tissues and should facilitate the interpretation of conventional toxicity and carcinogenicity studies in rats. Morphologic features of other selected rat liver lesions are also presented.

New Insights into Functional Aspects of Liver Morphology

The liver is structurally and functionally complex and has been considered second only to brain in its complexity. Many mysteries still exist in this heterogeneous tissue whose functional unit of the lobule has continued to stump morphologists for over 300 years. The primary lobule, proposed by Matsumoto in 1979, has been gaining acceptance as the functional unit of the liver over other conceptual views because it’s based on vessel architecture and includes the classic lobule as a secondary feature. Although hepatocytes comprise almost 80% of the liver, there are at least another dozen cell types, many of which provide “cross-talk” and play important functional roles in the normal and diseased liver. The distribution and functional roles of all cells in the liver must be carefully considered in both the analysis and interpretation of research data, particularly data in the area of genomics and “phenotypic anchoring” of gene expression results. Discoveries regarding the functional heterogeneity of the various liver cell types, including hepatocytes, hepatic stellate cells, sinusoidal endothelia, and Kupffer cells, are providing new insights into our understanding of the development, prevention and treatment of liver disease. For example, functional differences along zonal patterns (centrilobular or periportal) have been demonstrated for sinusoidal endothelium, Kupffer cells, and hepatocytes and can explain the gradients and manifestations of disease observed within lobules. Intralobular gradients of bile uptake, glycogen depletion, glutamine synthetase, and carboxylesterase by hepatocytes; widened fenestrations in centrilobular sinusoidal lining cells; and differences in the components of centrilobular extracellular matrix or function of Kupffer cells have been demonstrated. Awareness of the complexities and heterogeneity of the liver will add to a greater understanding of liver function and disease processes that lead to toxicity, cancer, and other diseases.

Diagnostic Criteria for Degenerative, Inflammatory, Proliferative Nonneoplastic and Neoplastic Liver Lesions in Medaka (Oryzias latipes): Consensus of a National Toxicology Program Pathology Working Group

Diagnostic criteria are presented for degenerative, inflammatory, nonneoplastic proliferative, and neoplastic lesions in the liver of medaka (Oryzias latipes), a small fish species frequently used in carcinogenesis studies. The criteria are the consensus of a Pathology Working Group (PWG) convened by the National Toxicology Program. The material examined by the PWG was from medaka exposed to N-nitrosodiethylamine for 28 days, removed to clean water, and sacrificed 4, 6, or 9 mo after initiation of exposure. Degenerative lesions included hepatocellular intracytoplasmic vacuolation, hepatocellular necrosis, spongiosis hepatis, hepatic cysts, and hepatocellular hyalinization. Inflammatory lesions consisted of granulomas, chronic inflammation, macrophage aggregates, and focal lymphocytic infiltration. Nonneoplastic proliferative lesions comprised foci of cellular alteration (basophilic focus, eosinophilic focus, vacuolated focus, and clear cell focus) and bile duct hyperplasia. Neoplastic lesions included hepatocellular adenoma, hepatocellular carcinoma, cholangioma, and cholangiocarcinoma. Two lesions composed mainly of spindle cells were noted, hemangiopericytoma and spindle cell proliferation. Rather than being an exhaustive treatment of medaka liver lesions, this report draws from the published literature on carcinogen-induced liver lesions in medaka and other fish species and attempts to consolidate lesion criteria into a simplified scheme that might be useful to pathologists and other researchers using medaka lesions for risk assessment or regulatory purposes.

Blood gene expression signatures predict exposure levels

To respond to potential adverse exposures properly, health care providers need accurate indicators of exposure levels. The indicators are particularly important in the case of acetaminophen (APAP) intoxication, the leading cause of liver failure in the U.S. We hypothesized that gene expression patterns derived from blood cells would provide useful indicators of acute exposure levels. To test this hypothesis, we used a blood gene expression data set from rats exposed to APAP to train classifiers in two prediction algorithms and to extract patterns for prediction using a profiling algorithm. Prediction accuracy was tested on a blinded, independent rat blood test data set and ranged from 88.9% to 95.8%. Genomic markers outperformed predictions based on traditional clinical parameters. The expression profiles of the predictor genes from the patterns extracted from the blood exhibited remarkable (97% accuracy) transtissue APAP exposure prediction when liver gene expression data were used as a test set. Analysis of human samples revealed separation of APAP-intoxicated patients from control individuals based on blood expression levels of human orthologs of the rat discriminatory genes. The major biological signal in the discriminating genes was activation of an inflammatory response after exposure to toxic doses of APAP. These results support the hypothesis that gene expression data from peripheral blood cells can provide valuable information about exposure levels, well before liver damage is detected by classical parameters. It also supports the potential use of genomic markers in the blood as surrogates for clinical markers of potential acute liver damage.

Examination of bone marrow, immunologic parameters and host susceptibility following pre- and postnatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

The effects of pre/postnatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on various immunological, bone marrow and host susceptibility assays were examined in B6C3F1 hybrid mice. Exposure was accomplished by maternal dosing on Day 14 of gestation and again on Days 1, 7, and 14 following birth, employing dosages of 0, 1.0, 5.0 or 15.0 micrograms/kg body weight. The 15.0 micrograms/kg dosage was lethal to 70% of the offspring with the remainder of that dosage group revealing overt toxicity. Bone marrow toxicity occurred in both the 15.0 and 5.0 micrograms/kg dosage groups as evidenced by bone marrow hypocellularity and depressed colony formation of macrophage-granulocyte progenitor cells and pleuripotent stem cells. Evidence was presented that depression of lymphoproliferative responses following mitogen stimulation in TCDD-immunosuppressed mice was due to a functional defect of lymphocyte activation rather than suppressor cell activity. Administration of either Listeria monocytogenes or syngeneic PYB6 tumor cells in mice exposed to relatively low levels of TCDD during pre- and postnatal development increased their susceptibility to either bacterial or tumor challenge.

Renal Lesions Induced by Ochratoxin A Exposure in the F344 Rat

Groups of 80 male and female F344 rats were exposed by gavage to ochratoxin A, a naturally occurring mycotoxin, at levels of 21, 70, and 210 μg/kg body weight for up to 2 years. Ochratoxin A induced nonneoplastic renal tubular epithelial changes consisting of cytoplasmic alteration, karyomegaly, degeneration, and cysts. Exposure-related renal tubular proliferative lesions included focal hyperplasia, tubular cell adenoma, and tubular cell carcinoma. Renal tubular cell adenoma occurred as early as 9 months in 1 high-dose male rat, and both adenomas and carcinomas were seen in males by 15 months. At the terminal sacrifice, renal tubular cell tumors were found in both male and female rats, but the response was more pronounced in the males. The incidence of renal tumors in the high-dose rats was the highest of any National Toxicology Program (NTP) study completed to date. In the high-dose males approximately one-third of the renal carcinomas developed metastases. This study demonstrates that ochratoxin A is a potent renal carcinogen in the F344 rat and suggests that contamination of feedstuff by this mycotoxin may pose a potential hazard to domestic animals and man.