Differences in drug sensitivity between two-dimensional and three-dimensional culture systems in triple-negative breast cancer cell lines.

Three-dimensional (3D) culture reflects tumor biology complexities compared with two-dimensional (2D) culture. Thus, 3D culture has attracted attention in cell biology studies including drug sensitivity tests. Herein, we investigated differences in anticancer drug sensitivities between 2D and 3D culture systems in triple-negative breast cancer (TNBC) cell lines. Thirteen TNBC cell lines were maintained in 2D and 3D cultures for 3 days before drug exposure. Cell morphology in the 3D culture was examined by phase-contrast microscopy. Sensitivities to epirubicin (EPI), cisplatin (CDDP), and docetaxel (DTX) were investigated by cell viability assay in both cultures and compared. The IC50s of all 3 drugs were significantly higher in the 3D culture than in the 2D culture in most cell lines. Those were correlated between the 2D and 3D cultures in EPI (R = 0.555) and CDDP (R = 0.955), but not in DTX (R = 0.221). Round spheroid-forming cells were more resistant to agents than grape-like types. In conclusion, 3D culture was more resistant to all 3 drugs than 2D culture in most TNBC cell lines. Sensitivity to CDDP was highly correlated between the 2D and 3D cultures, but not to DTX. 2D culture may be acceptable for sensitivity test for DNA-damaging agents.

Association of BRCA Mutations and BRCAness Status With Anticancer Drug Sensitivities in Triple-Negative Breast Cancer Cell Lines

BACKGROUND: Many triple-negative breast cancers (TNBCs) show impaired breast cancer susceptibility gene I (BRCA1) function, called BRCAness. BRCAness tumors may show similar sensitivities to anticancer drugs as tumors with BRCA1 mutations. In this study, we investigated the association of BRCA mutations or BRCAness with drug sensitivities in TNBC. METHODS: BRCAness was evaluated as BRCA1-like scores, using multiplex ligation-dependent probe amplification in 12 TNBC cell lines, including four with mutations. Sensitivities to docetaxel, cisplatin, and epirubicin were compared with BRCA mutations and BRCA1-like scores. Cisplatin sensitivity was examined in BRCA1 knockdown Michigan Cancer Foundation-7 cell lines. RESULTS: Eight and four cell lines had characteristics of BRCAness and non-BRCAness, respectively. The 50% inhibitory concentration of docetaxel was higher in BRCA mutant and BRCAness cell lines than their counterparts. BRCA1-like scores showed a weak positive correlation with docetaxel sensitivity (r = 0.377; P = 0.039). Regarding cisplatin, scores were lower in BRCA mutants and BRCAness tumors than their counterparts. A negative correlation was found between BRCA1-like scores and cisplatin sensitivity (r = -0.407; P = 0.013). No differences were found for epirubicin. BRCA1 gene knockdown increased the cisplatin sensitivity of Michigan Cancer Foundation-7 cells. CONCLUSIONS: BRCA1-like scores were associated with cisplatin sensitivity and docetaxel resistance. BRCA1-like score is hence a promising indicator for estimating drug sensitivities in TNBC.

ß-Tubulin Isoforms Related to Docetaxel Sensitivity in 2D and 3D Cultured TNBC Cell Lines.

BACKGROUND/AIM: We previously reported that the half maximal inhibitory concentration (IC50) values of cisplatin and epirubicin correlated in 13 triple-negative breast cancer (TNBC) cell lines between two-dimensional (2D) and three-dimensional (3D) culture methods. However, the IC50 values of docetaxel (DTX) did not correlate between the two culture methods. We hypothesized that this non-correlation is partly associated with differences in expression of the ß-tubulin isoform, the target molecule of DTX and in morphology depending on the culture method. MATERIALS AND METHODS: We investigated the expression levels of ß-tubulin isoforms by real-time polymerase chain reaction and morphology of spheroid formation in the 13 TNBC cell lines cultured using the 2D and 3D culture methods. RESULTS: Tubulin ß class I (TUBB) expression levels were negatively correlated with the IC50 value of DTX in the 2D culture method (R=-0.360), whereas tubulin ß class IIa (TUBB2a) expression levels were positively correlated in the 3D culture method (R=0.398). There was no significant difference in the expression levels of ß-tubulin isoforms between the 2D and 3D culture methods. The spheroids were classified morphologically into three types: round, mass, and grape-like. However, no clear association was found between DTX sensitivity and morphology. CONCLUSION: The non-correlation of the IC50 values of DTX between the 2D and 3D culture methods does not appear to be due to the changes in ß-tubulin isoforms. Morphology in the 3D culture method may play some role in drug sensitivity.

Beneficial effects of transdermal administration of tamoxifen on capsular contracture after breast implantation in murine models.

BACKGROUND AND AIM: Capsular contracture is the most common complication with smooth-type silicone implants. We investigated the preventive effect of an active metabolite of tamoxifen, 4-hydroxytamoxifen (4-OH TAM), on capsular contracture. METHODS: A silicone sheet was implanted into the back of 28 female ICR mice. Mixtures of gel with 0.2% 4-OH TAM and 0.1% 4-OH TAM were administered transdermally once a day for 4 weeks. Saline was administered to the control. After killing the mice, capsular thickness was measured in H&E-stained specimens. Estrogen receptor (ER), α-smooth muscle actin (α-SMA), and transforming growth factor-ß (TGF-ß) expressions were immunohistochemically investigated in the capsules. RESULTS: The capsule was thinner in the 0.2% 4-OH TAM gel group than in the control group (control, 0.1% 4-OH TAM gel, 0.2% 4-OH TAM gel: 52.8 ± 3.4 µm, 54.2 ± 6.8 µm, 46.4 ± 3.3 µm, respectively). ER was found in most fibroblasts of all samples. α-SMA expression in the capsule was significantly lower in the 4-OH TAM gel groups than in the control group (control = 70.0 ± 3.4%, 0.1% 4-OH TAM = 57.0 ± 3.4%, 0.2% 4-OH TAM = 49.4 ± 4.9%). TGF-ß expression was significantly reduced by the 4-OH TAM gel injections dose-dependently (control = 67.3 ± 2.2%, 0.1% 4-OH TAM = 52.4 ± 3.1%, 0.2% 4-OH TAM = 45.1 ± 2.4%). CONCLUSIONS: The transdermal administration of 0.1% and 0.2% 4-OH TAM gels inhibited capsule development. The inhibition of TGF-ß expression is a mechanism by which 4-OH TAM suppresses fibroblast growth, preventing capsular formation.

Involvement of oxidative stress in hepatocellular tumor-promoting activity of oxfendazole in rats.

The tumor-promoting effects of oxfendazole (OX), a benzimidazole anthelmintic, were investigated using a medium-term rat hepatocarcinogenesis model. Six-week-old male F344 rats received an intraperitoneal injection of N-diethylnitrosamine (DEN) and were given a powdered diet containing 0 or 500 ppm OX for 6 weeks from 2 weeks after DEN treatment. All animals were subjected to two-thirds partial hepatectomy 1 week after OX treatment. The numbers and areas of glutathione S-transferase placental form (GST-P)-positive foci were significantly increased in the livers of rats treated with OX, with concomitantly increased cell proliferation, compared with those in the livers of the DEN alone group. Quantitative real-time RT-PCR analysis revealed that OX induced not only mRNA expression of phase I enzymes Cyp1a1, Cyp1a2, but also Nrf2-regulated phase II enzymes such as Gpx2, Nqo1, Yc2, Akr7a3 and Gstm1, presumably due to an adaptive response against OX-induced oxidative stress. Reactive oxygen species production increased in microsomes isolated from the livers of OX-treated rats. Furthermore, OX enhanced oxidative DNA damage (as assessed by 8-hydroxydeoxyguanosine; 8-OHdG) and lipid peroxidation (as assessed by thiobarbituric acid-reactive substances; TBARS). These results suggest that administration of OX at a high dose and for a long term enhances oxidative stress responses, which may contribute to its tumor-promoting potential in rats.

Threshold dose of piperonyl butoxide that induces reactive oxygen species-mediated hepatocarcinogenesis in rats.

To determine the threshold dose of piperonyl butoxide (PBO) that induces hepatocellular tumor-promoting effects, reactive oxygen species (ROS) generation, and drug-metabolizing enzymes that protect against ROS generation, partial hepatectomized rats were fed diets containing 0, 0.015, 0.03, 0.06, 0.125, 0.25, or 0.5% PBO after an i.p. injection of N-diethylnitrosamine (DEN) to initiate hepatocarcinogenesis. Histopathologically, Glutathione S-transferase placental form (GST-P)-positive foci were significantly increased in a dose-dependent manner in rats given 0.25% PBO or higher. The formation of microsomal ROS in the liver was significantly increased in 0.25 and 0.5% PBO. Real-time RT-PCR showed that the expression of the CYP1A1, UDPGTr-2, and Mrp3 genes was significantly upregulated in rats given 0.03% PBO or higher. These results suggest that 0.25% is the threshold dose of PBO that induces ROS-mediated hepatocarcinogenesis in rats, although the CYP1A1 gene that is related to ROS generation and the UDPGTr-2 and Mrp3 genes that are involved in protection against ROS were induced in the livers of rats even at a PBO dose of 0.03%.

beta-Naphthoflavone enhances oxidative stress responses and the induction of preneoplastic lesions in a diethylnitrosamine-initiated hepatocarcinogenesis model in partially hepatectomized rats.

The tumour-promoting effects of beta-naphthoflavone (BNF), a novel aryl hydrocarbon receptor (AhR) agonist, were investigated using a medium-term hepatocarcinogenesis model in rats. Six-week-old male F344 rats received an intraperitoneal injection of N-diethylnitrosamine (DEN) at a dose of 200mg/kg body weight and were fed a diet containing 0% (basal diet), 0.5% or 1% BNF for 6 weeks from 2 weeks after DEN treatment. All animals were subjected to two-thirds partial hepatectomy 1 week after the BNF treatment. The number and area of glutathione S-transferase placental form (GST-P) positive foci significantly increased in the livers of rats treated with BNF with concomitantly increased cell proliferation compared to those in the livers of the DEN alone group. Global gene expression analysis and subsequent quantitative real-time reverse transcription-polymerase chain reaction revealed that BNF induced not only the 'AhR gene battery'Cyp1a1, Cyp1a2, Cyp1b1, Nqo1, Aldh3a1 and Ugt1a6 but also the transcription factor NF-E2-related factor 2 (Nrf2)-regulated genes such as Gstm1, Gpx2, Akr7a3 and Yc2 (and also Nqo1), presumably due to the adaptive response against BNF-triggered oxidative stress responses. Reactive oxygen species production increased in microsomes isolated from the livers of BNF-treated rats, and this enhancement was suppressed by the P450 inhibitor SKF-525A. Furthermore, BNF enhanced oxidative DNA damage and lipid peroxidation, estimated by the levels of 8-hydroxydeoxyguanosine (8-OHdG) and thiobarbituric acid-reactive substances. These results suggest that the administration of BNF at a high dose and over a long-term enhance oxidative stress responses which may contribute to its hepatocarcinogenic potential in rats.

Piperonyl butoxide activates c-Jun and ATF-2 in the hepatocytes of mice.

In order to clarify the possible mechanism of hepatocarcinogenesis induced by piperonyl butoxide, we attempted to identify the transcription factor activated by piperonyl butoxide in the male ICR mouse liver. Administration of 0.6% piperonyl butoxide for 24 h elevated the level of liver nuclear proteins that bind to an AP-1 consensus oligonucleotide, and these proteins demonstrated a supershift with the anti-c-Jun antibody. Additionally, immunoblot analysis revealed that piperonyl butoxide induced c-Jun phosphorylation within 8 h of administration, and phosphorylated ATF-2 was detected after 24 h of piperonyl butoxide treatment. Immunohistochemical analysis also demonstrated the presence of phosphorylated ATF-2 in the hepatocyte nuclei of mice fed with 0.6% piperonyl butoxide for 24 h. Furthermore, piperonyl butoxide induced ATF-2 phosphorylation in TLR-3, a mouse immortalized hepatocyte cell line. These results indicated that piperonyl butoxide activated c-Jun and ATF-2 in mouse hepatocytes during the early stage of hepatocarcinogenesis.

The possible mechanism of enhanced carcinogenesis induced by genotoxic carcinogens in rasH2 mice.

Microarray and RT-PCR analyses were performed for the transgene and Ras-related genes in forestomach squamous cell carcinomas (SCCs) induced by 7,12-dimethylbenz[a]anthracene (DMBA) in rasH2 mice; these results were compared with our previous molecular data of N-ethyl-N-nitrosourea-induced forestomach SCCs and urethane-induced lung adenomas in rasH2 mice. Overexpression of the transgene was detected in the DMBA-induced SCCs, suggesting that the transgene plays an important role in enhanced carcinogenesis in rasH2 mice. In addition, the mouse endogenous ras genes were up-regulated in the DMBA-induced SCCs, and are probably involved in the tumorigenesis of forestomach SCCs. Genes such as osteopontin, Cks1b, Tpm1, Reck, gelsolin, and amphiregulin that were commonly altered in these three different carcinogen-induced tumors may contribute to the development of tumors in rasH2 mice.

Effect of fenofibrate on oxidative DNA damage and on gene expression related to cell proliferation and apoptosis in rats.

To investigate the relationship between fenofibrate (FF) and oxidative stress, enzymatic, histopathological, and molecular biological analyses were performed in the liver of male F344 rats fed 2 doses of FF (Experiment 1; 0 and 6000 ppm) for 3 weeks and 3 doses (Experiment 2; 0, 3000, and 6000 ppm) for 9 weeks. FF treatment increased the activity of enzymes such as carnitine acetyltransferase, carnitine palmitoyltransferase, fatty acyl-CoA oxidizing system, and catalase in the liver. However, it decreased those of superoxide dismutase in the liver in both experiments. Increased 8-hydroxy-2'-deoxyguanosine levels in liver DNA and lipofuscin accumulation were observed in the treated rats of Experiment 2. In vitro measurement of reactive oxygen species (ROS) in rat liver microsomes revealed a dose-dependent increase due to FF treatment. Microarray (only Experiment 1) or real-time reverse transcription-polymerase chain reaction analyses revealed that the expression levels of metabolism and DNA repair-related genes such as Aco, Cyp4a1, Cat, Yc2, Gpx2, Apex1, Xrcc5, Mgmt, Mlh1, Gadd45a, and Nbn were increased in FF-treated rats. These results provide evidence of a direct or indirect relationship between oxidative stress and FF treatment. In addition, increases in the expression levels of cell cycle-related genes such as Chek1, Cdc25a, and Ccdn1; increases in the expression levels of cell proliferation-related genes such as Hdgfrp3 and Vegfb; and fluctuations in the expression levels of apoptosis-related genes such as Casp11 and Trp53inp1 were observed in these rats. This suggests that cell proliferation induction, apoptosis suppression, and DNA damage due to oxidative stresses are probably involved in the mechanism of hepatocarcinogenesis due to FF in rats.

Possible involvement of oxidative stress in piperonyl butoxide induced hepatocarcinogenesis in rats.

To clarify the possible mechanism of non-genotoxic hepatocarcinogenesis induced by piperonyl butoxide (PBO), male F344 rats were administered an i.p. injection of N-diethylnitrosamine (DEN) to initiate hepatocarcinogenesis. Two weeks later, the rats were administered a PBO-containing (0, 1, or 2%) diet for 6 weeks and subjected to a two-third partial hepatectomy 1 week later. After sacrificing them on week 8, their livers were histopathologically examined and analyzed for gene expression using a microarray and real-time RT-PCR. Reactive oxygen species (ROS) products were also measured using liver microsomes. Hepatocytes exhibited centrilobular hypertrophy and increased glutathione S-transferase placental form (GST-P) positive foci formation. ROS products increased significantly in liver microsomes. In the microarray analysis, the expressions of genes related to metabolism and oxidative stress - NAD(P)H dehydrogenase, quinone 1 (Nqo1), UDP-glucuronosyltransferase (UDPGTR-2), glutathione peroxidase 2 (Gpx2), glutathione reductase (GRx) - multidrug resistance associated protein 3 (Abcc3), and solute carrier family 7 (cationic amino acid transporter, y+ system) member 5 (Slc7a5) were up-regulated in the PBO group in comparison to the 0% PBO group; this was confirmed by real-time RT-PCR. Additionally, a significant up-regulation of stress response related genes such as CYP1A1 was observed in PBO-treated groups in real-time RT-PCR. HPLC analysis revealed that the level of 8-OHdG in the 2% PBO group was significantly higher than that in the 0% PBO group. This suggests that PBO has the potential to generate ROS via metabolic pathways and induce oxidative stress, including oxidative DNA damage, resulting in the induction of hepatocellular tumors in rats.

Thirteen-week repeated dose toxicity of Siraitia grosvenori extract in Wistar Hannover (GALAS) rats.

Siraitia grosvenori extract has been used as a food additive. As a part of the safety assessment of the extracts, a 13-week repeated dose toxicity study was performed in Wistar Hannover (GALAS) rats. Male and female rats were divided into five groups consisting of eight animals each and given diet containing 0%, 0.04%, 0.2%, 1%, and 5% of S. grosvenori extract for 13 weeks. During the experiment, no deaths were observed in any groups, and there were no remarkable changes in general appearance, body weight, food and water consumption, hematological and serum biochemical parameters, organ weight and histopathological findings between the control and treated groups. On the basis of these data, the no-observed-adverse effect level (NOAEL) of S. grosvenori extract in Wistar Hannover rats was considered to be 5% (2520 mg/kg/day in males and 3200 mg/kg/day in females) or more.

Enhancement of hepatocellular proliferative activity of kojic acid in mice by a simultaneous administration of ascorbic acid.

To examine the tumor modification activity of kojic acid (KA) by sodium ascorbic acid (AA), 5-week-old male ICR mice were administered intraperitoneally with N-diethylnitrosamine (DEN) as an initiation treatment. Two weeks after the initiation treatment, animals were fed basal diet containing 0 (Group 1: DEN alone) or 3% KA (Group 3: DEN+KA), drinking water containing 5,000 ppm AA (Group 2: DEN+AA) or 3% KA and 5,000 ppm AA (Group 4: DEN+KA+AA) for 6 weeks. One week after the administration of KA and/or AA, all mice were subjected to two-thirds partial hepatectomy. At the end of the experimental period, all surviving mice were sacrificed and removed the liver. The liver weights of the Groups 3 and 4 were significantly increased, and the number of proliferating cell nuclear antigen positive hepatocytes and the gene expressions of Ccnc, Ccnd1, Ercc and Cyp7a1 were significantly increased in the Group 4, as compared to the Group 1. These results of the present study suggest that AA enhances the hepatocellular proliferative activity of KA in mice.

Molecular pathological analysis for determining the possible mechanism of piperonyl butoxide-induced hepatocarcinogenesis in mice.

Piperonyl butoxide (PBO), alpha-[2-(2-butoxyethoxy)ethoxy]-4,5-methylene-dioxy-2-propyltoluene, is widely used as a synergist for pyrethrins. In order to clarify the possible mechanism of non-genotoxic hepatocarcinogenesis induced by PBO, molecular pathological analyses consisting of low-density microarray analysis and real-time reverse transcriptase (RT)-PCR were performed in male ICR mice fed a basal powdered diet containing 6000 or 0 ppm PBO for 1, 4, or 8 weeks. The animals were sacrificed at weeks 1, 4, and 8, and the livers were histopathologically examined and analyzed for gene expression using the microarray at weeks 1 and 4 followed by real-time RT-PCR at each time point. Reactive oxygen species (ROS) products were also measured using liver microsomes. At each time point, the hepatocytes of PBO-treated mice showed centrilobular hypertrophy and increased lipofuscin deposition in Schmorl staining. The ROS products were significantly increased in the liver microsomes of PBO-treated mice. In the microarray analysis, the expression of oxidative and metabolic stress-related genes--cytochrome P450 (Cyp) 1A1, Cyp2A5 (week 1 only), Cyp2B9, Cyp2B10, and NADPH-cytochrome P450 oxidoreductase (Por) was over-expressed in mice given PBO at weeks 1 and 4. Fluctuations of these genes were confirmed by real-time RT-PCR in PBO-treated mice at each time point. In additional real-time RT-PCR, the expression of Cyclin D1 gene, key regulator of cell-cycle progression, and Xrcc5 gene, DNA damage repair-related gene, was significantly increased at each time point and at week 8, respectively. These results suggest the possibility that PBO has the potential to generate ROS via the metabolic pathway and to induce oxidative stress, including oxidative DNA damage, resulting in the induction of hepatocellular tumors in mice.

Concurrent administration of ascorbic acid enhances liver tumor-promoting activity of kojic acid in rats.

We previously found that administration of ascorbic acid (AA) enhances the liver tumor-promoting activity of kojic acid (KA) in mice. To examine the reproducibility of these results in rats and the underlying mechanism of this effect, we employed a two-stage liver carcinogenesis model using male F344 rats. Two weeks after initiation with diethylnitrosamine (DEN), the animals received a diet containing 2% KA and drinking water with or without 5,000 ppm AA for a period of 7 weeks. A DEN-alone group was also established as a control. One week after the commencement of the administration, the animals were subjected to two-thirds partial hepatectomy. At the end of the experiment, the livers were analyzed immunohistochemically, and the mRNA expression level and extent of lipid peroxidation were measured. AA treatment enhanced the KA-induced tumor-promoting activity in terms of the number and area of liver cell foci that were positive for glutathione-S-transferase placental form. AA coadministration increased the number of hepatocytes positive for proliferating cell nuclear antigen and inversely decreased the number of TUNEL-positive cells. However, the increased level of thiobarbituric acid reactive substances resulting from KA treatment was suppressed by coadministration of AA. Gene expression analyses using low-density microarrays and real-time RT-PCR showed that coadministration of AA resulted in upregulation of genes related to cell proliferation and downregulation of those involved in apoptosis and/or cell cycle arrest. These results indicate that the concerted effects of AA on cell proliferation and apoptosis/cell cycle arrest probably through its antioxidant activity are involved in this enhancement.

Modifying effect of Siraitia grosvenori extract on piperonyl butoxide-promoted hepatocarcinogenesis in rats.

To examine the possible modifying effect of the extract of Siraitia grosvenori (SGE), a naturally occurring antioxidative agent, on piperonyl butoxide (PBO)-promoted hepatocarcinogenesis, male F344 rats were administered a single intraperitoneal injection of N-diethylnitrosamine (DEN) as an initiator followed by administration of a diet containing 2% PBO for 7 weeks with or without SGE (1,000 ppm) in the drinking water. To enhance cellular proliferation, all animals underwent two-thirds partial hepatectomy 1 week after the commencement of PBO administration. Pretreatment with SGE was also applied to the PBO + SGE group for 2 weeks prior to DEN initiation. Liver immunohistochemistry revealed that although the PBO-mediated increase in the number of glutathione S-transferase placental form (GST-P)-positive foci and proliferating cell nuclear antigen-positive cells remained unaltered with SGE coadministration, the area of the GST-P-positive foci was increased. On the contrary, real-time RT-PCR showed that coadministration of SGE increased hepatic GST and glutathione peroxidase (GSH-Px) antioxidant activities and mRNA expression levels of the phase II enzymes that are known to be transcriptionally up-regulated through the Nrf 2-Keap1-antioxidant responsive element (ARE) as well as the phase III enzymes. Furthermore, measurement of thiobarbituric acid-reactive substances showed a decrease in lipid peroxidation by SGE coadministration. The results suggest that SGE may exert hepatic antioxidant activity by up-regulating the genes under the control of the Nrf 2-Keap1-ARE transcriptional machinery; however, this activity was neither effective nor sufficient for suppression of PBO-promoted early hepatocarcinogenesis.

Possible involvement of oxidative stress in fenofibrate-induced hepatocarcinogenesis in rats.

To clarify whether oxidative stress is involved in the development of hepatocellular preneoplastic foci induced by fenofibrate (FF), a peroxisome proliferator-activated receptor alpha agonist, male F344/N rats were fed a diet containing 6,000, 3,000, or 0 ppm of FF for 13 weeks after N-diethylnitrosamine initiation. Two-third partial hepatectomy was performed 1 week after the FF treatment. Histopathologically, the number of hepatocellular altered foci significantly increased in the FF-treated groups with a concomitant increase in the number of hepatocytes positive for anti-Ki-67 antibody, but the number and area of glutathione S-transferase placental form (GST-P)-positive foci decreased in these groups, as compared to those in the controls. Microarray analysis or quantitative real-time reverse transcription-polymerase chine reaction demonstrated the significant up-regulations of Aco and Cyp4a1 (genes related to lipid metabolism); Gpx2, Yc2, Cat, Cyp2b15, and Ugt1a6 (metabolic oxidative stress-related genes); Apex1, Mgmt, Xrcc5, Nbn, and Gadd45a (DNA repair-related genes); and Ccnd1 (cell cycle-related genes) in the FF-treated groups, and the significant down-regulations of Cyp1a2, Gsta2, Gstm2, and Gstm3 (phase I or II metabolism-related genes); Mlh1 and Top1 (DNA repair-related genes); and Cdkn1a, Cdkn1b, Chek2, and Gadd45b (cell cycle/apoptosis-related genes) in these rats. FF-treatment increased the activity of enzymes such as carnitine acetyltransferase, carnitine palmitoyltransferase, fatty acyl-CoA oxidizing system, and catalase in the liver, but not superoxide dismutase in the liver. In addition, 8-OHdG level in liver DNA, lipofuscin deposition in hepatocytes, and in vitro reactive oxygen species production in microsomes significantly increased due to FF treatment. These results suggest that oxidative stress is involved in the development of FF-induced hepatocellular preneoplastic foci in rats.

Gene expression analyses of the liver in rats treated with oxfendazole.

The effect of oxfendazole (OX), a benzimidazole anthelmintic, on hepatic gene expression was investigated in the liver of rats as a preliminary study to elucidate the possible mechanism of its non-genotoxic hepatocarcinogenesis. The liver from a male F344/N rat given a diet containing 500 ppm of OX for 3 weeks was examined by global gene expression analysis in comparison with an untreated rat. Microarray analysis revealed that phase I and phase II detoxifying enzymes were up-regulated in an OX-treated rat. In addition to these genes, the expressions of several upregulated genes related to xenobiotic metabolism and oxidative stress [e.g. Cyp1a1; NAD(P)H dehydrogenase, quinone 1 (Nqo1); glutathione peroxidase 2 (Gpx2); glutathione S-transferase Yc2 subunit (Yc2)], were confirmed by real-time reverse transcription polymerase chain reaction (RT-PCR). Furthermore, rats were administered 500 or 1,000 ppm of OX for 9 weeks, and the effect of OX on oxidative stress responses was evaluated by real-time RT-PCR along with conventional toxicological assays, including lipid peroxidation (thiobarbituric acid-reactive substance; TBARS). A longer treatment period and/or a higher dose of OX tended to increase the gene expressions of not only phase I (Cyp1a1 and Cyp1a2) but also phase II (Nqo1, Gpx2, Yc2, and Akr7a3) drug metabolizing enzymes. Toxicological parameters, such as TBARS, serum aspartate aminotransferase (AST), and serum alkaline phosphatase (ALP), showed slight but significant increases after treatment with OX for 9 weeks. These results indicate that OX elicits adaptive responses against oxidative stress in the liver and suggest that the imbalance in redox status might be one of the factors triggering the initial step of OX-induced non-genotoxic carcinogenesis in the liver of rats.

Carcinogenic susceptibility of rasH2 mice to troglitazone.

To evaluate the carcinogenicity of troglitazone in rasH2 mice, 7-week-old male and female rasH2 mice were fed a diet containing 0, 3,000 or 6,000 ppm troglitazone for 26 weeks. An increased tendency in the incidence of vascular tumors was observed in females of the 6,000 ppm group. The preliminary analysis using a high-density oligonucleotide microarray on a splenic hemangiosarcoma of a high dose female that could be obtained as a fresh sample showed that several genes related to the ras/MAPK pathway activation, angiogenesis, cell cycle and cell multiplication were up-regulated. In addition, most of the genes up-regulated were confirmed by the reverse transcriptase-polymerase chain reaction (RT-PCR). These results may suggest that the carcinogenic susceptibility of rasH2 mice to troglitazone is relatively low and up-regulations of the ras/MAPK pathway and angiogenesis-related genes are probably involved in the production of splenic hemangiosarcomas in rasH2 mice given troglitazone.

Possible involvement of oxidative stress in dicyclanil-induced hepatocarcinogenesis in mice.

Our previous study suggested the possibilities that dicyclanil (DC), a nongenotoxic carcinogen, produces oxidative stress in the liver of the two-stage hepatocarcinogenesis model of mice and the stress induced probably causes secondary oxidative DNA damage. However, clear evidences demonstrating the relationship between DC-induced hepatocarcinogenesis, oxidative stress, and oxidative DNA damage have not been obtained. To clarify the relationship, further investigations were performed in the liver of the partially hepatectomized (PH) mice maintained on diet containing 1,500 ppm of DC for 13 and 26 weeks after intraperitoneal injection of dimethylnitrosamine (DMN). Significant increases in mRNA expressions of some metabolism- and oxidative stress-related genes with a formation of gamma-glutamyltranspeptidase (GGT) positive foci were observed in the DMN + DC + PH group by the treatment of DC for 13 and 26 weeks. The levels of 8-hydroxy-deoxyguanosine (8-OHdG) in the liver DNA also significantly increased in mice of the DMN + DC + PH group at weeks 13 and 26 and mice given DC alone for 26 weeks. The in vitro measurement of reactive oxygen species (ROS) generation from the mouse liver microsomes showed a significant increase of ROS production in the presence of DC. These results suggest that DC induces oxidative stress which is probably derived from its metabolic pathway, partly, and support our previous speculation that oxidative stress plays one of the important roles in the DC-induced hepatocarcinogenesis in mice.