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Archives of Toxicology (v.85, #12)

Most cited articles: ethanol-induced hepatotoxicity, anticarcinogenic effects of polyphenolic compounds in tea, dose–response modeling, novel roles of epoxide hydrolases and arsenic-induced suicidal erythrocyte death by H. M. Bolt; J. G. Hengstler (pp. 1485-1489).

Highlight report: the bisphenol A controversy by H. M. Bolt; J. D. Stewart (pp. 1491-1492).

Selenium: an element with two faces by H. M. Bolt; R. Marchan; J. G. Hengstler (pp. 1493-1494).

Muller’s Nobel lecture on dose–response for ionizing radiation: ideology or science? by Edward J. Calabrese (pp. 1495-1498).

In his Nobel Prize Lecture of December 12, 1946, Hermann J. Muller argued that the dose–response for radiation-induced germ cell mutations was linear and that there was “no escape from the conclusion that there is no threshold”. However, assessment of correspondence between Muller and Curt Stern 1 month prior to his Nobel Prize Lecture reveals that Muller knew the results and implications of a recently completed study at the University of Rochester under the direction of Stern, which directly contradicted his Nobel Prize Lecture. This finding is of historical importance since Muller’s Nobel Lecture gained considerable international attention and is a turning point in the acceptance of the linearity model in risk assessment for germ cell mutations and carcinogens.

Keywords: Linearity; Threshold; Hermann J. Muller; Nobel Prize; Risk assessment; X-rays; Ionizing radiation

Evaluation of biocompatible dispersants for carbon nanotube toxicity tests by Jin Sik Kim; Kyung Seuk Song; Ji Hyun Lee; Il Je Yu (pp. 1499-1508).

Dispersion is one of the key obstacles to evaluating the in vitro and in vivo toxicity of carbon nanotubes (CNTs), as the aggregation or agglomeration of CNTs in culture media or vehicles complicates the interpretation of the toxicity test results. Thus, to test the dispersion of CNTs in biocompatible solutions, 5 known biocompatible dispersants were selected that are widely used for nanomaterial toxicity evaluation studies. Single-wall nanotubes (SWCNTs) and multi-wall nanotubes (MWCNTs) were both dispersed in these dispersants and their macrodispersion evaluated using a light absorbance method. The dispersion stability of the dispersed SWCNTs and MWCNTs was also evaluated for 16 weeks, plus the dispersants were tested for their innate toxicity using trypan blue dye exclusion, lactate dehydrogenase (LDH) leakage, and neutral red assays. All the dispersants were found to be biocompatible in the cytotoxicity tests when compared with a positive control of 2% Triton X-100. In the dispersion tests, 0.02, 0.1, and 0.5% MWCNTs and SWCNTs were diluted in the respective dispersants. Distilled water and dimethylsulfoxide (DMSO) both showed a poor macrodispersion of only 1–13% for the various CNT concentrations. In 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), the 0.02 and 0.1% MWCNTs showed a macrodispersion of 11 and 74%, respectively, while the 0.02 and 0.1% SWCNTs showed a macrodispersion of 15 and 16%, respectively. In 0.5% bovine serum albumin (BSA), the 0.02, 0.1, and 0.5% MWCNTs showed a very good macrodispersion of 32, 53, and 70%, respectively, yet the 0.02% SWCNTs only showed a macrodispersion of 17%. In 1% Tween 80, the 0.02–0.5% SWNCTs exhibited a good macrodispersion of 27–81%, whereas the 0.02–05% MWCNTs only showed a macrodispersion of 13–23%. The dispersion stability of the CNTs during 16 weeks was in the following descending order of BSA, Tween 80, DPPC, and DMSO for the MWCNTs and BSA, DPPC, Tween 80, and DMSO for the SWNCTs. Thus, appropriate dispersants are proposed according to the type of CNT, experiment concentration, and treatment duration. Also, it is suggested that the dispersibility, dispersion stability, and biocompatibility of the selected dispersant should all be confirmed before a toxicity evaluation.

Keywords: Carbon nanotubes (CNTs); Dispersion; Aggregation; Agglomeration; Biocompatible; Single-wall nanotubes (SWCNTs); Multi-wall nanotubes (MWCNTs); Dispersants

Rat H9c2 cardiac myocytes are sensitive to arsenite due to a modest activation of transcription factor Nrf2 by Daigo Sumi; Takahiko Sasaki; Hideki Miyataka; Seiichiro Himeno (pp. 1509-1516).

The mechanism underlying the hepatotoxicity induced by arsenic exposure is well investigated. However, little is known about the detailed mechanisms of arsenic-induced cardiotoxicity or cardiac factors involved in high sensitivity to arsenicals in spite of the fact that arsenic trioxide, which is used to treat acute promyelocytic leukemia, causes cardiotoxicity. Here, we show that rat H9c2(2-1) cardiac myocytes exhibit high sensitivity to inorganic arsenite (As(III)) as compared with rat-derived four cell lines (liver epithelial TRL1215 cells, kidney epithelial NRK-52E cells, PC12 phechromocytoma cells and C6 glioma cells). Furthermore, we found a lower steady-state level of glutathione and glutamyl-cysteine ligase (GCL) in H9c2(2-1) cells compared with TRL1215 cells, resulting in an increase in arsenic accumulation. In addition, we detected that the up-regulation of GCL and multi-drug resistance-associated protein (MRP) caused by As(III) was extremely low in H9c2(2-1) cells compared with TRL1215 cells. It is known that Nrf2, which regulates GCL and MRP expression, plays an important role in the protection of cells from arsenicals. We investigated the participation of Nrf2 in the difference of sensitivity to arsenicals between H9c2(2-1) and TRL1215 cells and found that Nrf2 was clearly activated by As(III) exposure in TRL1215 cells but only poorly activated in H9c2(2-1) cells. Considering these results together, we propose that modest activation of Nrf2 during exposure to As(III) in H9c2(2-1) cardiac myocytes leads to reduced ability to metabolize and excrete arsenic.

Keywords: Arsenic; Nrf2; Glutathione; MRP

Evaluation of the cytotoxic and inflammatory potential of differentially shaped zinc oxide nanoparticles by Boon Chin Heng; Xinxin Zhao; Eng Chok Tan; Nurulain Khamis; Aarti Assodani; Sijing Xiong; Christiane Ruedl; Kee Woei Ng; Joachim Say-Chye Loo (pp. 1517-1528).

Zinc oxide (ZnO) nanoparticles have wide-ranging applications in a diverse array of industrial and consumer products, from ceramic manufacture and paint formulation to sunscreens and haircare products. Hence, it is imperative to rigorously characterize the health and safety aspects of human exposure to ZnO nanoparticles. This study therefore evaluated the cellular association, cytotoxic and inflammatory potential of spherical and sheet-shaped ZnO nanoparticles (of approximately the same specific surface area ≈30 cm²/g) on mouse and human cell lines (RAW-264.7 and BEAS-2B respectively), as well as with primary cultures of mouse bone marrow-derived dendritic cells (DC). The WST-8 assay demonstrated dose-dependent effects on the cytotoxicity of spherical and sheet-shaped ZnO nanoparticles on both RAW-264.7 and BEAS-2B cells, even though there was no significant effect of shape on the cytotoxicity of ZnO nanoparticles. There was however higher cellular association of spherical versus sheet-shaped ZnO nanoparticles. Measurement of reactive oxygen species (ROS) with the 2′,7′-dichlorfluorescein-diacetate (DCFH-DA) assay indicated up to 4-folds increase in ROS level upon exposure to ZnO nanoparticles, but there was again no significant difference between both ZnO nanoparticle shapes. Exposure of primary dendritic cells to ZnO nanoparticles upregulated expression of CD80 and CD86 (well-known markers of DC activation and maturation) and stimulated release of pro-inflammatory cytokines—IL-6 and TNF-α, thus pointing to the potential of ZnO nanoparticles in inducing inflammation. Hence, our study indicated that ZnO nanoparticles can have potential detrimental effects on cells even at dosages where there are little or no observable cytotoxic effects.

Keywords: Cytotoxicity; Inflammation; Nanoparticle; Oxide; Zinc

Silver nanoparticles induce apoptosis and G2/M arrest via PKCζ-dependent signaling in A549 lung cells by Young Sook Lee; Dong Woon Kim; Young Ho Lee; Jung Hwa Oh; Seokjoo Yoon; Mi Sun Choi; Sung Kyu Lee; Ji Won Kim; Kyuhong Lee; Chang-Woo Song (pp. 1529-1540).

The use of silver nanoparticles is one of the fastest growing product categories in the nanotechnology industry, with a focus on antimicrobial activity. However, thus far, toxicity data for silver nanoparticles are limited. In this study, we investigated the cytotoxic effects of silver nanoparticles (Ag NPs) and the pathway by which they affect A549 lung epithelial cells. The effects of Ag NPs on cell survival, cell cycle progression, and mRNA and protein alterations of selected cell cycle- and apoptosis-related genes were studied using formazan dye and LDH release assays, flow cytometric analysis, semi-quantitative RT-PCR, and Western blot analysis. Ag NPs reduced cell viability, increased LDH release, and modulated cell cycle distribution through the accumulation of cells at G2/M and sub-G1 phases (cell death), with a concurrent decrease in cells at G1. Ag NP treatment increased Bax and Bid mRNA levels and downregulated Bcl-2 and Bcl-w mRNAs in a dose-dependent manner. Furthermore, Ag NPs altered the mRNA levels of protein kinase C (PKC) family members. In particular, ectopic overexpression of PKCζ led to the enhancement of cellular proliferation and reduced sensitivity to Ag NPs in A549 cells. Together, these results suggest that Ag NPs induce strong toxicity and G2/M cell cycle arrest by a mechanism involving PKCζ downregulation in A549 cells.

Keywords: Silver nanoparticles (Ag NPs); G2/M arrest; Apoptosis; Protein kinase C (PKC) ζ

Okadaic acid and dinophysis toxin 2 have differential toxicological effects in hepatic cell lines inducing cell cycle arrest, at G0/G1 or G2/M with aberrant mitosis depending on the cell line by J. A. Rubiolo; H. López-Alonso; F. V. Vega; M. R. Vieytes; L. M. Botana (pp. 1541-1550).

Okadaic acid is one of the toxins responsible for the human intoxication known as diarrhetic shellfish poisoning, which appears after the consumption of contaminated shellfish. The main diarrhetic shellfish poisoning toxins are okadaic acid, dinophysistoxin-1, -2, and -3. In vivo, after intraperitoneal injection, dinophysistoxin-2 is approximately 40% less toxic than okadaic acid in mice. The cytotoxic and genotoxic effect of okadaic acid varies very significantly in different cell lines, so similar responses could be expected for dinophysistoxin-2. In order to determine whether this was the case, we studied the effect of okadaic acid and dinophysistoxin-2 in two hepatic cell lines (HepG2 and Clone 9). The cytotoxicity of these toxins, as well as their effects on the cell cycle and its regulation on both cell lines, were determined. Okadaic acid and dinophysistoxin-2 resulted to be equipotent in clone 9 cultures, while okadaic acid was more potent than dinophysistoxin-2 in HepG2 cell cultures. Both toxins had opposite effects on the cell cycle; they arrested the cell cycle of clone 9 cells in G2/M inducing aberrant mitosis while arresting the cell cycle of HepG2 in G0/G1. When the effect of the toxins on p53 subcellular distribution was studied, p53 was detected in the nuclei of both cell types. The effect of the toxins on the gene expression of cyclins and cyclin-dependent kinases was different for both cell lines. The toxins induced an increase in gene expression of cyclins A, B, and D in clone 9 cells while they induced a decrease in cyclins A and B in HepG2 cells. They also induced a decrease in cyclin-dependent kinase 1 in HepG2 cells.

Keywords: Okadaic acid; Dinophysis toxin-2; Differential toxicity; Cell cycle

A new Phaseolus vulgaris lectin induces selective toxicity on human liver carcinoma Hep G2 cells by Evandro Fei Fang; Wen Liang Pan; Jack Ho Wong; Yau Sang Chan; Xiu Juan Ye; Tzi Bun Ng (pp. 1551-1563).

We describe here the purification and characterization of a new Phaseolus vulgaris lectin that exhibits selective toxicity to human hepatoma Hep G2 cells and lacks significant toxicity on normal liver WRL 68 cells. This polygalacturonic acid–specific lectin (termed BTKL) was purified from seeds of P. vulgaris cv. Blue tiger king by liquid chromatography techniques. The 60-kDa dimeric lectin showed strong and broad-spectrum hemagglutinating activity toward human, rabbit, rat, and mouse erythrocytes. Bioinformatic analysis unveils substantial N-terminal sequence similarity of BTKL to other Phaseolus lectins. Among a number of tumor cells tested, BTKL exhibits potent anti-Hep G2 activity which is associated with (1) induction of DNA fragmentation, (2) production of apoptotic bodies and chromatin condensation, (3) triggering of cell apoptosis and necrosis, and (4) depolarization of mitochondrial membrane (low ΔΨm). Furthermore, BTKL could induce inducible nitric oxide synthase (iNOS) expression and subsequent nitric oxide production in vitro in mouse macrophages, which may contribute to its antitumor activity. In addition, BTKL could bring about a significant dose-dependent increase in the production of mRNAs of proinflammatory cytokines including interleukin-1 beta, interleukin-2, tumor necrosis factor alpha, and interferon-gamma. In sum, the antitumor activity and mechanism of BTKL provided here suggest that it has potential therapeutic value for human liver cancer.

Keywords: Liver cancer; Apoptosis; Lectin; Nitric oxide; Cytokine

In vivo assessment of antiemetic drugs and mechanism of lycorine-induced nausea and emesis by Sascha Kretzing; Getu Abraham; Bettina Seiwert; Fritz Rupert Ungemach; Ute Krügel; Jens Teichert; Ralf Regenthal (pp. 1565-1573).

Lycorine is the main alkaloid of many Amaryllidaceae and known to cause poisoning with still unknown mechanisms. Longer lasting toxicological core symptoms of nausea and emesis may become a burden for human and animal patients and may result in substantial loss of water and electrolytes. To optimise the only empirical symptomatic antiemetic drug treatment at present, it is important to elucidate the causative involved targets of lycorine-induced emesis. Therefore, in the current study, we have tested the actions of a various antiemetic drugs with selective receptor affinities on lycorine-induced nausea and emesis in vivo in dogs. Beagle dogs were pre-treated in a saline vehicle-controlled crossover and random design with diphenhydramine, maropitant, metoclopramide, ondansetron or scopolamine prior lycorine administration (2 mg/kg subcutaneously). In vivo effects were assessed by a scoring system for nausea and emesis as well as by the number and lag time of emetic events for at least 3 h. Moreover, plasma pharmacokinetic analysis was carried out for ondansetron before and after lycorine injection. The data show that histaminergic (H₁), muscarinic and dopaminergic (D₂) receptors are presumably not involved in lycorine-induced emetic effects. While ondansetron significantly reduced the number of emetic events, lycorine-induced emesis was completely blocked by maropitant. Only ondansetron also significantly decreased the level of nausea and was able to prolong the lag time until onset of emesis suggesting a preferential participation of 5-HT₃ receptors in lycorine-induced nausea. Thus, it is the first in vivo report evidencing that predominantly neurokinin-1 (NK₁) and to a lesser extent 5-hydroxytryptamine 3 (5-HT₃) receptors are involved in lycorine-induced emesis facilitating a target-oriented therapy.

Keywords: Lycorine; Alkaloid; Amaryllidaceae; Diphenhydramine; Maropitant; Metoclopramide; Ondansetron; Scopolamine; Toxicity; Mechanism; Emesis; Dogs

Variation in the internalization of differently sized nanoparticles induces different DNA-damaging effects on a macrophage cell line by Mingyi Zhang; Juan Li; Gengmei Xing; Rui He; Wei Li; Yan Song; Haili Guo (pp. 1575-1588).

Although researchers have expended considerable effort on studying the cytotoxicity of nanomaterials, it is possible that there has been insufficient attention paid to their genotoxic potential. Here, we describe a test model that we have developed to evaluate the DNA-damaging effects of negatively charged nanoparticles of different sizes. We compared the DNA damaging effect induced by nanoparticles of various sizes and found that the effect is closely associated with the internalization pattern of the particles. Macrophage cell line RAW 264.7 cells were incubated with carboxylated polystyrene beads (COOH–PBs) ranging in size from 30 to 500 nm. Size-dependent DNA damage was detected, and the lesion induced by two carboxylated fullerene particles confirmed this observation. Confocal microscopy revealed that the entry pathways of these COOH–PBs shifted from direct penetration to endocytosis with increasing particle size, followed by changes in subcellular localization. Subsequent deposition of 30-nm COOH–PBs in the cytosol led to a reduction of Zn²⁺ and Mg²⁺ content in the nucleus and an increased p53 level in the whole cell rather than in nucleus, while localization of 50- and 100-nm COOH–PBs in acidic vesicles induced p53 accumulation in both types of extracts. Based on these results, we assume that the damage resulted from a disruption of the balance between DNA damage and repair.

Keywords: DNA damage; Entry pathway; Carboxylated nanoparticles; Size-dependency; RAW 264.7

Characterization of spontaneous cell death in monolayer cultures of primary hepatocytes by Mathieu Vinken; Elke Decrock; Tatyana Doktorova; Eva Ramboer; Elke De Vuyst; Tamara Vanhaecke; Luc Leybaert; Vera Rogiers (pp. 1589-1596).

Monolayer cultures of primary hepatocytes, isolated from freshly removed livers, represent widely used in vitro tools in the area of liver physiology and pathology, pharmacology and toxicology. However, a major shortcoming of these systems is that they cope with dedifferentiation, which is accompanied by spontaneous cell death. The goal of the present study was to elucidate the mechanisms that drive the process of self-generated cell demise in primary hepatocyte cultures. For this purpose, isolated rat hepatocytes were cultivated under conventional conditions, and the occurrence of apoptosis and necrosis was monitored during 4 days by performing a set of acknowledged cell death assays. These included examination of cell morphology by light microscopy, quantification of apoptotic and necrotic cell populations by Hoechst 33342 and propidium iodide in situ staining, assessment of apoptotic and necrotic activities by measuring caspase 3-like activity and extracellular leakage of lactate dehydrogenase, and studying the expression of apoptosis regulators through immunoblot analysis. In essence, two cell death peaks were observed, namely shortly after cell seeding and in the final stages of the cultivation period, both involving apoptotic and necrotic actions. The outcome of this study not only sheds new light onto the molecular processes that underlie spontaneous cell death in primary hepatocyte cultures, but also opens perspectives for the establishment of strategies to increase cell survival in these popular in vitro systems.

Keywords: Primary hepatocytes; Spontaneous apoptosis; Secondary necrosis

Role of endogenous hydrogen sulfide on renal damage induced by adriamycin injection by Heloísa Della Coletta Francescato; Evelyn Cristina Santana Marin; Fernando de Queiroz Cunha; Roberto Silva Costa; Cleonice Giovanini Alves da Silva; Terezila Machado Coimbra (pp. 1597-1606).

A single injection of adriamycin (ADR) induces marked and persistent proteinuria in rats that progress to glomerular and tubulointerstitial lesions. It has been shown that ADR-induced nephrotoxicity is mediated, at least in part, by oxidative stress that lead to inflammation. Endogenous hydrogen sulfide (H₂S) is synthesized from l-cysteine and is an important signaling molecule in inflammation. This study evaluates the effect of DL-propargylglycine (PAG), an inhibitor of endogenous H₂S formation, on the evolution of renal damage induced by ADR. The rats were injected i.p. with 0.15 M NaCl or PAG (50 mg/kg) 2 h after ADR injection (3.5 mg/kg). Control rats were injected with 0.15 M NaCl or PAG only. Twenty hours urine samples were collected for albuminuria and creatinine measurements on days 1 and 14 after saline or ADR injections and on days 2 and 15 blood samples were collected to measure plasma creatinine, then the rats were killed. The kidneys were removed for H₂S formation evaluation, renal lipid peroxidation and glutathione levels, and histological and immunohistochemical analysis. On day 2 after ADR injection the rats presented increase in oxidative stress associated with neutrophils and macrophages influx in renal tissue. On day 15 the rats also presented increased desmin expression at glomerular edge and vimentin in cortical tubulointerstitium, as well as albuminuria. All these alterations were reduced by PAG injection. The protective effect of PAG on ADR nephrotoxicity was associated to decreased H₂S formation and to restriction of oxidative stress and inflammation in the renal cortex.

Keywords: Adriamycin; DL-propargylglycine; Inflammation; Renal damage

In vivo microdialysis and electroencephalographic activity in freely moving guinea pigs exposed to organophosphorus nerve agents sarin and VX: analysis of acetylcholine and glutamate by John C. O’Donnell; John H. McDonough; Tsung-Ming Shih (pp. 1607-1616).

Organophosphorus nerve agents such as sarin (GB) and VX irreversibly inhibit acetylcholinesterase, causing a buildup of acetylcholine (ACh) in synapses and neuromuscular junctions, which leads to excess bronchial secretions, convulsions, seizures, coma, and death. Understanding the unique toxic characteristics of different nerve agents is vital in the effort to develop broad spectrum medical countermeasures. To this end, we employed a repeated measure multivariate design with striatal microdialysis collection and high-performance liquid chromatography analysis to measure changes in concentrations of several neurotransmitters (ACh, glutamate, aspartate, GABA) in the same samples during acute exposure to GB or VX in freely moving guinea pigs. Concurrent with microdialysis collection, we used cortical electrodes to monitor brain seizure activity. This robust double multivariate design provides greater fidelity when comparing data while also reducing the required number of subjects. No correlation between nerve agents’ propensity for causing seizure and seizure-related lethality was observed. The GB seizure group experienced more rapid and severe cholinergic toxicity and lethality than that of the VX seizure group. Seizures generated from GB and VX exposure resulted in further elevation of ACh level and then a gradual return to baseline. Glutamate levels increased in the GB, but not in the VX, seizure group. There were no consistent changes in either aspartate or GABA as a result of either nerve agent. These observations reinforce findings with other nerve agents that seizure activity per se contributes to the elevated levels of brain ACh observed after nerve agent exposure.

Keywords: Acetylcholine; Acetylcholinesterase; Choline; Electroencephalogram; γ-Aminobutylic acid (GABA); Glutamate; Guinea pig; In vivo microdialysis; Nerve agents; Organophosphorus compounds; Sarin; Seizure activity; VX

Genotoxic effects of the cyanobacterial hepatotoxin cylindrospermopsin in the HepG2 cell line by Alja Štraser; Metka Filipič; Bojana Žegura (pp. 1617-1626).

The cyanobacterial alkaloid cylindrospermopsin (CYN) is being increasingly identified in drinking water supplies worldwide. It is a potent protein synthesis inhibitor and causes human intoxications and animal mortality. The few genotoxicity studies available indicate that CYN is genotoxic, generally implying that it is pro-genotoxic. We evaluated CYN genotoxicity in the human hepatoma cell line, HepG2, analyzing the induction of DNA strand breaks, with the alkaline comet assay, and micronuclei (MNi), nuclear bud (NBUD), and nucleoplasmic bridge (NPB) formation, with the cytokinesis block micronucleus (CBMN) assay. In addition, changes in the expression of genes involved in the response to DNA damage (P53, CDKN1A, GADD45α, and MDM2) and genes presumably involved in CYN metabolism (genes from the Cytochrome P450 family: CYP1A1 and CYP1A2) were determined, using quantitative real-time PCR. Non-cytotoxic concentrations of CYN induced increased DNA damage after 12 and 24 h of exposure and increased the frequency of MNi, NBUDs, and NPBs after 24 h exposure. Moreover, CYN up-regulated the expression of the CYP1A1 and CYP1A2 genes. Although no changes in the expression of the P53 tumor-suppressor gene were found, CYN up-regulated the expression of the P53 downstream-regulated genes CDKN1A, GADD45α, and MDM2. Our results provide new evidence that CYN is genotoxic and strongly suggest that it needs to be considered in the human health risk assessment.

Keywords: Cylindrospermopsin; DNA damage; Micronucleus; Nuclear bud; Nucleoplasmic bridge; Gene expression

Identification of biomarkers of chemically induced hepatocarcinogenesis in rasH2 mice by toxicogenomic analysis by Han-Jin Park; Jung-Hwa Oh; Se-Myo Park; Jae-Woo Cho; Young Na Yum; Sue Nie Park; Do-Young Yoon; Seokjoo Yoon (pp. 1627-1640).

Toxicogenomic approaches have been applied to chemical-induced heptocarcinogenesis rodent models for the identification of biomarkers of early-stage hepatocarcinogenesis and to help clarify the underlying carcinogenic mechanisms in the liver. In this study, we used toxiciogenomic methods to identify candidate biomarker genes associated with hepatocarcinogenesis in rasH2 mice. Blood chemical, histopathologic, and gene expression analyses of the livers of rasH2 mice were performed 7 and 91 days after the administration of the genotoxic hepatocarcinogens 2-acetylaminofluorene (AAF) and diethylnitrosoamine (DEN), the genotoxic carcinogen melphalan (Mel), and the nongenotoxic noncarcinogen 1-naphthylisothiocynate (ANIT). Histopathologic lesions and a rise in accompanying serum marker levels were found in the DEN-treated rasH2 mice, whereas no neoplastic lesions were observed in the rasH2 mice. However, biological functional analysis using Ingenuity Pathways Analysis (IPA) software revealed that genes with comparable molecular and cellular functions were similarly deregulated in the AAF- and DEN-treated rasH2 mice. We selected 68 significantly deregulated genes that represented a hepatocarcinogen-specific signature; these genes were commonly deregulated in both the AAF- and DEN-treated rasH2 mice on days 7 and 91. Hierarchical clustering analysis indicated that the expression patterns of the selected genes in the hepatocarcinogen (AAF and DEN) groups were distinctive from the patterns in the control, Mel, and ANIT groups. Biomarker filter analysis using IPA software suggested that 28 of the 68 signature genes represent promising candidate biomarkers of cancer. Quantitative real-time PCR analysis confirmed that the deregulated genes, which exhibited sustained up- and down-regulation up to day 91, are likely involved in early-stage hepatocarcinogenesis. In summary, the common and significant gene expression changes induced by AAF and DEN may reflect early molecular events associated with hepatocarcinogenesis, and these “signature” genes may be useful as biomarkers of hepatocarcinogenesis in mice.

Keywords: Biomarker; Hepatocarcinogenesis; rasH2 mice; Toxicogenomics; 2-acetylaminofluorene; Diethylnitrosoamine

Erratum to: Anti-apoptotic, anti-inflammatory and immunomodulatory activities of 3,3′-diselenodipropionic acid in mice exposed to whole body γ-radiation by A. Kunwar; P. P. Bag; S. Chattopadhyay; V. K. Jain; K. I. Priyadarsini (pp. 1641-1643).

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Archives of Toxicology (v.85, #12)