Toxicologic Pathology

Experimental ovarian carcinogenesis has been investigated in inbred and hybrid strains of mice and induced by a diversity of mechanisms including X-irradiation, oocytotoxic xenobiotic chemicals, ovarian grafting to ectopic or orthotopic sites, neonatal thymectomy, mutant genes reducing germ cell populations, and aging. Disruptions in the function of graafian follicles by a variety of mechanisms results in a spectrum of ovarian proliferative lesions, including tumors. The findings in mutant and genetically engineered mice support the concept of a secondary (hormonally mediated) mechanism of ovarian tumorigenesis in mice associated with sterility. Multiple pathogenic factors that either destroy or diminish the numbers of graafian follicles in the ovary result in decreased sex hormone secretion, especially estradiol-17 β, leading to a compensatory overproduction of pituitary gonadotrophins, particularly LH, which places the mouse ovary at an increased risk for developing tumors in chronic studies. The intense proliferation of ovarian surface epithelium and stromal (interstitial) cells with the development of unique tubular adenomas in response to sterility does not appear to have a counterpart in the ovaries of adult human females.

The peroxisome proliferators represent an important group of hepatic carcinogens in rodents that act via the nuclear receptor PPARα. The primary role of PPARα in mediating this response had led to the further characterization of potential events downstream that likely enable the carcinogenic response, including increased peroxisomal fatty acid beta oxidation and the modulation of hepatocellular replication and death, either generally or in preneoplastic lesions. A cooperative role of Kupffer cell activation has been proposed to function in the modulation of hepatocellular proliferation in rodent liver by peroxisome proliferators, but data that confirm or refute this proposal are mixed. Presently there is no evidence that links the Kupffer cell activation by peroxisome proliferators directly to the development of liver tumors. There are marked species differences in susceptibility to peroxisomal proliferation, and active investigation concerning the molecular basis of these differences continues.

Benzene is a potent human leukemogen but the mechanism underlying benzene-induced leukemia remains an enigma due to a number of questions regarding the requirement of extraordinarily long exposure, a relatively low incidence of leukemia for genotoxicity of metabolites and a narrow dose range for leukemogenicity over marrow aplasia (overdoses tend to result in marrow aplasia). Moreover, there were previous controversies as to whether the cell cycle is upregulated or suppressed by the benzene exposure. Subsequently, it was found that the cell cycle is suppressed, but how leukemia develops under such suppression of hemopoiesis remains to be clarified. These questions were fortunately resolved with much effort. Benzene exposure was found to induce the expression of p21, an interlocking counterdevice for cell cycle: due to p53 upregulation, thereby inducing the immediate suppression of the kinetics of hemopoietic progenitors followed by the prominent suppression of hemopoiesis. Intermittent benzene exposure (i.e., cessation of exposure during weekends, for example) allowed an immediate recovery from marrow suppression after terminating exposure, which induced continuous oscillatory changes in marrow hemopoiesis. Benzene-induced leukemia was chiefly due to such an oscillatory change in hemopoiesis, which epigenetically developed leukemia more than 1 year later. The mechanisms of benzene-induced leukemogenicity seem to differ between wild-type mice and mice lacking p53. For p53 knockout mice, DNA damage such as weak mutagenicity or chromosomal damage was retained, and such damage induced consequent activation of proto-oncogenes and related genes, which led cells to undergo further neoplastic changes. In contrast, for wild-type mice carrying the p53 gene, a marked oscillatory change in the cell cycle of the stem cell compartment seems to be important. Compatible and discriminative gene expression profiling between the p53 knockout mice and wild-type mice was observed after benzene exposure by microarray analyses.

In the Federal Register of January 10, 2000 (65 FR 1399), FDA published a draft guidance entitled “Photosafety Testing.” The notice gave interested persons an opportunity to submit comments. As a result of the comments, certain sections of the guidance were reworded to improve clarity. A final guidance was published in May 2003. The final guidance further emphasizes that a flexible approach can be used to address adverse photoeffects and that specific assays are not required. Moreover, it encourages the development of methods that can efficiently be used to evaluate human safety. The guidance describes a consistent, science-based approach for testing of topically and systemically administered drug products.

Hepatotoxicity characterized by microvesicular steatosis (MVS) is characterized by an abnormal accumulation of numerous small cytoplasmic lipid droplets in hepatocytes. Fulminant or progressive cases of microvesicular steatosis may lead to liver failure and death. Experimentally, short-chain carboxylic acids are known to induce microvesicular steatosis. The identification of gene changes that correlate with MVS concomitant with biochemical and histological indices could provide a better understanding of how this toxicity occurs as well as biomarkers that could be used to avoid this toxicity in the future. Sprague—Dawley rats were dosed days with cyclopropane carboxylic acid (CPCA) a short-chain fatty acid that can induce microvesicular steatosis, and with butyrate, a short chain fatty acid that served as a negative control. CPCA initiated microvesicular steatosis while butyrate did not. In addition, CPCA inhibited beta-oxidation in a concentration-dependent manner in vitro and caused a reduction in mitochondrial respiration ex vivo; no inhibition was evident with butyrate. Microarray results showed that gene expression changes with CPCA resulted in regulation of genes involved in beta-oxidation, as well as other genes associated with mitochondrial function. Overall, these results support altered hepatic mitochondrial function as a mechanism of the toxicity induced by a short-chain fatty acid and may provide potential biomarkers for this toxicity.

The discovery that 3-hydroxy-3-methyglutaryl coenzyme A reductase was a rate-determining step in the biosynthesis of cholesterol led to the discovery of inhibitors of this enzyme. To support the development of these agents (statins) as potential hypocholesterolemic drugs, a variety of preclinical studies were conducted in several animal species. Not unexpectedly due to the central role played by mevalonic acid and its products including cholesterol in development and maintenance of cellular homeostasis, administration of high dosage levels of these agents led to the expression of a broad variety of adverse effects in many different tissues. Using the tools of toxicologic pathology and classical risk assessment, these varied toxicities were evaluated by many groups relative to the conditions of use in human therapy and a perspective was developed on potential human risk. These approaches of mechanism-based risk assessment predicted that most of the adverse effects observed in animals would not be seen under conditions of human use and supported the successful introduction of one of the most important classes of human medicines.

Magnetic resonance imaging (MRI) has enjoyed enormous clinical success since the first demonstration of the method more than 30 years ago. An increasing number of pharmaceutical manufacturers seeking new biomarkers for assessing drug efficacy and toxicity are turning to MRI. A specific application of MRI promises to revolutionize pathology for the basic scientist in the same way MRI has forever altered the standard of care in the clinical domain. More specifically, this application is the use of magnetic resonance microscopy (MRM) in conjunction with new staining methodologies that now make MRM routinely available to the widest range of investigators.

Digital microscopy, a comprehensive integration of digital imaging and light microscopy, can assist the pathologist to observe, acquire, record, share, analyze, and manage pathology image data. To lead the activity for establishing new generation digital microscopy capacity, novel concepts and strategies of digital pathology information flow and digital pathology platform were designed to integrate personal digital pathology microscopy workstations and other pathology imaging modalities with centralized data storage/management. In addition, a strategy for Web-enabled interactive telepathology that would permit global capacity was designed. A novel concept of high content pathology was also created to develop an automated tissue microscopy imaging and screening approach. These new concepts, strategies, and approaches guided the development and implementation of a digital pathology platform, a telepathology platform, and automated tissue slide imaging capacity. Digital microscopy photography is now able to replace photographic film in toxicologic pathology. Digital pathology and telepathology platforms can provide a networked environment for multisite, global team participation. Our practice also ascertained the central value of digital microscopy which can provide innovative quantitative pathology information and data mining capability with various imaging biomarkers via advanced digital image processing and pathology informatics; these are now the focus of ongoing development.

Public concerns have been raised in recent years over the possible adverse effects that may result from exposure to chemicals in the environment that have the potential to interfere with the normal function of the endocrine system in wildlife and humans (“endocrine disrupters”). Regulations have been established that require the testing of pesticides used in food crops and drinking water contaminants, for estrogenicity and other hormonal activities. In the United States, the U.S. EPA proposed the Endocrine Disrupter Screening Program, which consists of a Tier 1 screening battery of tests that is designed to identify chemicals capable of interacting with various hormonal systems, and different Tier 2 testing assays that are designed to verify and broaden the Tier 1 results. We identify 2 main problems with this approach: (1) the fact that the developmental stages that are the most susceptible to endocrine disruption are not represented in the screening tier, mainly because developmental effects tend to be latent, and there is no way to economically screen in developing models; and (2) the expense to screen each chemical to be included in this program. Thus, the need arises for an accurate, rapid, and cost effective method for assessing the potential endocrine activity of multiple chemicals during development. We hypothesize that the largely latent developmental effects of some endocrine disruptors are preceded by immediate changes in gene expression in the embryo and fetus. Therefore, an approach to assess the potential estrogenic (and other steroid hormonal) activity of different compounds is to identify those patterns of gene expression elicited in a tissue/organ exposed to these particular classes of chemicals. In this paper, the potential utility of such an approach for screening and better understanding of mechanism of action for specific chemicals with endocrine disrupter activities is presented, using as an example chemicals with estrogenic activity.

It is almost ironic that one of the major organs that serves to maintain the “internal milieux” by secretion of various toxic agents, can itself become injured in the process. The pattern of morphologic renal injury is nonspecific and can involve any of the components of the kidney, although the injury and subsequent morphologic changes are most commonly noted in the tubules and/or interstitium. Of course, unless the drug/toxin is commonly or regularly noted to be associated with tubular and/or interstitial injury, the association of the drug with the renal changes may be missed and the correlation may not necessarily identify causation. For example, if a drug is associated with a renal injury in a given individual, it may be quite difficult to prove that the drug is the cause of the injury. This scenario is somewhat reminiscent of the test question—is it “true-true-related,” or “true-true-unrelated”? Sometimes it is only by the accrual of a great many examples or correlations, and or dissection of the pathophysiology, can it be shown that the drug is directly related to the observed morphologic (and subsequent clinical) injury. Renal changes induced by chemicals can affect the tubules, interstitium or both. This review of chemically induced nephropathy in humans considers acute tubular necrosis, interstitial nephritis , and tubulointerstitial nephritis or nephropathy. Because the tubules and the interstitium are so intimately related, injury to 1 of these 2 components may eventually lead to injury of the other, resulting in tubulointerstitial disease.

To explore for practical thresholds for DNA-reactive carcinogens in rat liver carcinogenicity, we have conducted a series of exposure-response studies using 2 well-studied hepatocarcinogens, 2-acetylaminofluorene (AAF) and diethylnitrosamine (DEN). Findings with AAF, including as yet unpublished experiments, are reviewed here and related to DEN observations. In these studies, we have administered exact intragastric doses during an initiation segment (IS) of 12—16 weeks followed in some experiments by phenobarbital (PB) as a liver tumor promoter for 24 weeks to enhance manifestation of initiation. The cumulative doses (CD) of AAF at the end of ISs ranged from 0.094 to 282.2 mg/kg. Our findings for AAF in the IS can be summarized as follows: (1) the earliest parameter to be affected with administration of low doses was the appearance of DNA adducts (around 4 weeks), followed at higher doses by cell proliferation; (2) formation of DNA adducts was nonlinear, with a no-observed effect level (NOEL) at a CD of 0.094 mg/kg and a plateau at higher doses (94.1 mg/kg); (3) cytotoxicity (necrosis) showed a NOEL at a CD of 28.2 mg/kg; (4) compensatory hepatocellular proliferation showed a NOEL at a CD of 28.2 mg/kg and was supralinear at a high CD (282.2 mg/kg); (5) formation of preneoplastic hepatocellular altered foci (HAF) showed a NOEL at a CD of 28.2 mg/kg, and was supralinear at a high CD (282.2 mg/kg); (6) a NOEL (CD 28.2 mg/kg) was found for tumor development and the exposure—response was supralinear. We interpret these findings to reflect practical thresholds for hepatocellular initiating effects of AAF and exaggerated responses at high-exposures doses, as also found for DEN. Thus, mechanisms of carcinogenesis can differ between low and high doses.