In vitro model systems have recently been developed to investigate the toxicity of chemical, microbial and physical agents in normal human tissues and cells from many of the major tissue sites of high cancer incidence. Pathobiological endpoints used in these studies include alterations in incorporation rates of precursors into DNA, RNA and protein, in the clonal growth rate of cultured cells and in DNA structure (e.g. single-strand breaks, DNA-protein crosslinks and chemical-DNA adducts) and the induction of differentiation, chromosomal and karyotypic abnormalities, mutations and neoplastic transformation. These systems have been used to study a variety of complex mixtures and individual substances, including cigarette smoke components such as benzo[a]pyrene and N-nitrosamines, formaldehyde, fecapentaenes, asbestos, and nickel and chromium ions. In addition, increasing awareness of the role of oncogenes in human carcinogenesis has led to studies involving transfection experiments with oncogenes and hepatitis B viral genes in normal human cells.

Asbestos↗

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The relationship between [Ca2+]i and cell death using an in vivo model: a study using the ced-1 mutant strain of C. elegans.

The ced-1 mutant of the free-living nematode, Caenorhabitis elegans, was used to study cell injury and cell death in relation to changes in intracellular ionized calcium ([Ca2+]i). This animal, which is being genetically characterized, may prove to be extremely useful for certain toxicologic studies because of its small size, optical transparency, rapid generation time, and the morphologic and genetic data currently available. During the development of this animal, 131 of 1,090 ultimate somatic cells undergo programmed cell death. Using mutagenesis techniques, several genes responsible for this death have been identified. In this study, we have taken advantage of the ced-1 mutant in which dead cells accumulate, as they cannot be phagocytized and removed. Although changes in [Ca2+]i have been studied in relation to cell injury and cell death, observations have been essentially restricted to in vitro monolayer cultures because of the methodology involved. To study the relationship between changes in [Ca2+]i and injury in vivo, we selected this animal model for further study and report here the morphological changes following the effects of ionomycin treatment in relation to increases of [Ca2+]i and cell death as measured using the fluorescent probes Fluo-3/AM and propidium iodide, respectively. The technique of confocal laser scanning microscopy is ideally adapted to such measurements in these living animals, and the results can be readily correlated with those made with Nomarski differential interference contrast microscopy as well as with transmission electron microscopy. The results support previous in vitro observations and show that early increases of [Ca2+]i accompany early reactions to injury. Furthermore, the results also show that changes in this small invertebrate metazoan parallel those seen in mammalian systems, including human. Thus, the current study indicates that ced-1 C. elegans can potentially serve as an in vivo model not only for evaluating the possible temporal relationship of [Ca2+]i elevation with cell death but also for evaluating the [Ca2+]i elevation observed in relation to other phenomena and in evaluating toxic agents.

Animals↗

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Characterization of a renal epithelial cell model of apoptosis using okadaic acid and the NRK-52E cell line.

Apoptotic cell death plays an important role in the pathogenesis of renal tubular epithelial damage and repair following tubular injury. Presently, the cellular factors involved in regulating apoptotic pathways in the kidney are unknown. To address the possibility that protein phosphorylation may regulate apoptosis in kidney cells, okadaic acid (OKA), a specific inhibitor of protein phosphatases 1A and 2A, was tested for its morphologic and biochemical effects on normal rat kidney epithelial cells (NRK-52E) in culture. As revealed by the DNA-specific stain DAPI, nuclei of cells treated with 1.0 microM okadaic acid contained irregular clumps of dense chromatin. Additional morphologic alterations typical of apoptosis were apparent within 2 hr after treatment with 1.0 microM OKA, including marked cellular rounding, cytoplasmic condensation, and cytoplasmic blebs. Ultrastructurally, 1.0 microM OKA caused cytoplasmic bleb formation, cellular fragmentation, condensation of heterochromatin into clumps, and segregation of nucleoli. At this stage, the cytoplasmic fragments and blebs contained many normal mitochondria. The attached, rounded cells also effectively excluded propidium iodide, demonstrating maintenance of membrane integrity despite pronounced morphologic alterations. A 2-fold increase in intracellular free Ca2+ was apparent 90 min after treatment with 1.0 microM okadaic acid. Transverse alternating field electrophoresis revealed the appearance of large DNA fragments of approximately 300-kbp. The appearance of these 300-kbp fragments correlated temporally with the observed elevation in intracellular calcium and the onset of morphologic alterations. However, preloading cells with EGTA-AM, an intracellular calcium chelator, obliterated the calcium elevation and had no effect on OKA-induced morphology, DNA fragmentation, or cell death. Detectable internucleosomal fragmentation occurred much later than the onset of morphologic changes (24-hr treatment time) and did not correlate with elevations in cellular calcium. These studies support the hypothesis that during apoptosis, chromatin condensation reflects chromatin cleavage at nuclease-sensitive sites between hexameric rosettes. These results also suggest that morphologic and nuclear alterations in the pathway of OKA-induced apoptosis occur independent of observed increases in intracellular calcium.

Animals↗

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Cell death is alive and well.

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Animals↗

PubMedDate not supplied

The pathways of cell death: oncosis, apoptosis, and necrosis.

The pathways and identification of cell injury and cell death are of key importance to the practice of diagnostic and research toxicologic pathology. Following a lethal injury, cellular reactions are initially reversible. Currently, we recognize two patterns, oncosis and apoptosis. Oncosis, derived from the Greek word "swelling," is the common pattern of change in infarcts and in zonal killing following chemical toxicity, e.g., centrilobular hepatic necrosis after CC14 toxicity. In this common reaction, the earliest changes involve cytoplasmic blebbing, dilatation of the endoplasmic reticulum (ER), swelling of the cytosol, normal or condensed mitochondria, and chromatin clumping in the nucleus. In apoptosis, the early changes involve cell shrinkage, cytosolic shrinkage, more marked chromatin clumping, cytoplasmic blebbing, swollen ER on occasion, and mitochondria that are normal or condensed. Following cell death, both types undergo postmortem changes collectively termed "necrosis." In the case of oncosis, this typically involves broad zones of cells while, in the case of apoptosis, the cells and/or the fragments are often phagocytized prior to their death by adjacent macrophages or parenchymal cells. In either case, the changes converge to a pattern that involves mitochondrial swelling, mitochondrial flocculent densities and/or calcification, karyolysis, and disruption of plasmalemmal continuity. The biochemical mechanisms of cell death are currently under intense study, particularly concerning the genes involved in the process. Pro-death genes include p53, the ced-3/ICE proteases, and the Bax family. Anti-death genes include ced-9/Bcl-2 and the adenovirus protein EIB. It is clear that ion deregulation, particularly that of [Ca2+]i plays an important role in cell death following either apoptosis or oncosis. Genetic evidence strongly indicates that activation of proteases is an important step, possibly very near to the point where cell death occurs.

Animals↗

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A personal perspective on the past, present, and future of Toxicologic Pathology.

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Animals↗

PubMedDate not supplied

Interspecies differences in renal localization of cyclooxygenase isoforms: implications in nonsteroidal antiinflammatory drug-related nephrotoxicity.

Cyclooxygenase (COX) exists in 2 related but unique isoforms: one is constitutive (COX-1) and functions in normal cell physiology, and the other is inducible (COX-2) and is expressed in response to inflammatory stimuli. Nonsteroidal antiinflammatory drugs (NSAIDs) cause renal toxicity following inhibition of renal cyclooxygenases. Humans and animals exhibit differences in susceptibility to NSAID-related renal toxicity, which may be associated with differences in expression of 1 or both isoforms of COX in the kidney. In this study, we evaluated COX-1 and COX-2 expression in the kidneys of mixed-breed dogs, Sprague-Dawley rats, cynomolgus monkeys, and humans. In addition, the effect of volume depletion on renal COX expression was investigated in rats, dogs, and monkeys. COX expression was evaluated using 1 or more of the following procedures: reverse transcriptase polymerase chain reaction, in situ hybridization, and immunohistochemistry. We demonstrated that both COX isoforms are expressed in the kidneys of all species examined, with differences in the localization and level of basal expression. COX-1 is expressed at high levels in the collecting ducts and renal vasculature of all species and in a small number of papillary interstitial cells in rats, monkeys, and humans. Basal levels of COX-2 are present in the maculae densa, thick ascending limbs, and papillary interstitial cells in rats and dogs and in glomerular podocytes and small blood vessels in monkeys and humans. COX-2 expression is markedly increased in volume-depleted rats and dogs but not monkeys. These results indicate that significant interspecies differences exist in the presence and distribution of COX isoforms, which may help explain the difference in species susceptibility to NSAID-related renal toxicity.

Adult↗

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The nomenclature of cell death: recommendations of an ad hoc Committee of the Society of Toxicologic Pathologists.

The last several years have seen considerable confusion regarding the terms "apoptosis" and "necrosis" in pathology. This situation prompted the Society of Toxicologic Pathologists to charter the Committee on the Nomenclature of Cell Death, which was charged with making recommendations about the use of the terms "apoptosis" and "necrosis" in toxicity studies. The Committee recommends use of the term "necrosis" to describe findings comprising dead cells in histological sections, regardless of the pathway by which the cells died. The modifiers "apoptotic" and "oncotic" or "mixed apoptotic and oncotic" are recommended to specify the predominant morphological cell death pathway or pathways, when appropriate. Other standard modifiers, indicating the lesion distribution and severity, may also be used in conjunction with these. "Individual cell necrosis" (also known as "single cell necrosis") may be either of the apoptotic, oncotic, or mixed types. In many cases, more traditional terms such as "coagulation necrosis" may be used to convey a meaning similar to oncotic necrosis. It is important that pathologists use terms that accurately and concisely convey the level of information appropriate to the study's needs. Furthermore, toxicologic pathologists should actively help to disseminate these recommendations to other biologists and to regulatory authorities.

Animals↗

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Biochemical and ultrastructural changes in teleost liver following subacute exposure to PCB.

The response of the channel catfish liver to subacute exposure of polychlorinated biphenyls was evaluated using electron microscopic and biochemical techniques. After 21 days, treated fish displayed elevated microsomal enzyme activities. Morphologically, the liver produced several patterns of alteration involving the endoplasmic reticulum (ER). Structural alterations included an increase in tubular smooth ER, production of parallel stacks of smooth ER showing continuity with rough ER, and membranous whorls. Biochemical and morphologic findings were correlated in exposed livers, and the relationship of these findings to similar studies in other species of fish is discussed.

Animals↗

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Allelic frequency of a p53 polymorphism in human lung cancer.

p53 is a tumor suppressor gene that is mutated in diverse tumor types. Here we report the frequencies of common polymorphic variants at codon 72 of the p53 gene in germline DNA of lung cancer cases and controls as determined by a polymerase chain reaction strategy. The observed allelic distribution was found to be significantly different between African-Americans and Caucasians in this U.S. population. The frequency of polymorphic variants was similar in lung cancer cases and controls after adjustment for race. However, among lung cancer patients the proline variant at codon 72 was in excess in adenocarcinoma patients by comparison with other histologies.

Adenocarcinoma↗

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Comparative toxicity of polychlorinated biphenyl and polybrominated biphenyl in the rat liver: light and electron microscopic alterations after subacute dietary exposure.

The comparative toxicity of polychlorinated biphenyl (PCB) and polybrominated biphenyl (PBB) in livers was studied in male Holtzman rats. Four-week-old animals were fed at dietary levels of 0, 5, 50, or 500 ppm for 5 weeks and then sacrificed. The mean liver-body weight ratios of the 50 and 500 ppm groups were increased. Histopathologic examination of the livers revealed fatty degenerative change associated with both compounds. This change was more marked at 500 than at 50 ppm. Various sized lamellar cytoplasmic inclusions were detected in livers of animals fed 500 ppm of either compound. However, the inclusions were more numerous in the PBB-treated rats. Several animals fed 50 ppm PBB had a few inclusions. In rats that received 500 ppm PBB, hypertrophic degenerative hepatocytes were present around the central veins. On the periphery of this change there were occasionally multinucleated hepatocytes. Electron microscopic examination at a dose level of 5 ppm in both the PCB and PBB groups showed a slight proliferation of smooth endoplasmic reticulum (SER), a moderate increase of lipid droplets and some liposomes, and a marked proliferation of Golgi condensing vesicles containing lipoprotein particles. A decreased number of mitochondria and lysosomes was also observed. At 50 ppm, similar but more marked ultrastructural alterations were seen. In addition, an increased number of branched and cup-shaped profiles of mitochondria and a decreased number of Golgi condensing vesicles containing lipoprotein particles were observed. Concentric membranous cytoplasmic whorls were encountered only in the 50 ppm PBB-treated rats. At 500 ppm the number of mitochondria decreased in both groups. There was also a marked increase in the number of SER and liposomes concomitant with a decreased number of Golgi condensing vesicles containing lipoprotein granules. Membranous whorls were also present in the 500 ppm groups.

Animals↗