Satoshi Uwagawa

Circulating microRNAs, possible indicators of progress of rat hepatocarcinogenesis from early stages.

MicroRNAs (miRNAs), a class of small noncoding RNAs that regulate gene expression at the posttranscriptional level, are believed promising biomarkers for several diseases as well as a novel target of drugs, including cancer. In particular, miRNAs might allow detection of early stages of carcinogenesis. The present study was conducted to provide concrete evidence using chemical-induced hepatocarcinogenesis in rat as a model. We thereby observed aberrant fluctuation of circulating miRNAs in the serum of rats not only with neoplastic lesions such as hepatocellular adenoma (HCA) and hepatocellular carcinoma (HCC), but also with preneoplastic lesions, such as foci of hepatocellular alteration (FHA). Additional qRT-PCR analysis revealed gradual elevation of some circulating miRNAs (i.e., let-7a, let-7f, miR-34a, miR-98, miR-331, miR-338 and miR-652) with progress of hepatocarcinogenesis. Interestingly, increased levels of let-7a, let-7f and miR-98 were statistically significant even in the serum of rats at very early stages. These findings provide the first evidences that circulating miRNAs have the potential to predict carcinogenesis at earlier stages, preneoplastic lesions than with previous biomarkers and that they might be utilized to monitor the progress of tumor development.

Detailed low-dose study of 1,1-B is(p-chlorophenyl)-2,2,2-trichloroethane carcinogenesis suggests the possibility of a hormetic effect

To obtain information on the effects of nongenotoxic carcinogens at low doses for human cancer risk assessment, the carcinogenic potential of the organochlorine insecticide, 1,1‐bis(p‐chlorophenyl)‐2,2,2‐trichloroethane (DDT), in the liver was assessed in F344 rats. In experiment 1, 240 male animals, 21 days old, were administered 0, 0.5, 1.0, 2.0, 5.0, 20, 100 and 500 ppm DDT in the diet for 16 weeks. Experiment 2 was conducted to elucidate the carcinogenic potential of DDT at lower levels using 180 rats given doses of 0, 0.005, 0.01, 0.1, 0.2 and 0.5 ppm. The livers of all animals were immunohistochemically examined for expression of glutathione S‐transferase placental form (GST‐P), putative preneoplastic lesions. Quantitative values for GST‐P‐positive foci in the liver were increased dose‐dependently in rats given 20 ppm DDT and above with statistical significance as compared with the concurrent control value. In contrast, doses of 0.005 and 0.01 ppm were associated with a tendency for decrease below the control value, although not significantly. Western blotting analysis show that cytochrome P‐450 3A2 (CYP3A2) protein expression tended to decrease at 0.005 and 0.01 ppm, a good correlation being observed with the change in the number of GST‐P‐positive foci. These findings suggest that a DDT hepatocarcinogenicity may show nonlinear response, that is, hormetic response at low doses. Furthermore, since CYP3A2 protein expression appears to be important for the effects of phenobarbital and the α‐isomer of benzene hexachloride, mRNAs for IL‐1 receptor type 1 (IL‐1R1) and TNF‐α receptor type 1 (TNFR1) whose ligands have roles not only in downregulating CYP3A2 expression but also in inducing antiproliferative effect or apoptosis in hepatocyte were examined. Increase was observed at low doses of DDT. Oxidative stress in liver DNA, assessed in terms of 8‐hydroxydeoxyguanosine as a marker, was also decreased. These findings suggest that the possible hormetic effect that was observed in our detailed low‐dose study of DDT carcinogenesis, although not statistically significant, may be linked to levels of oxidative stress and proinflammatory cytokines.

Case Study: An Evaluation of the Human Relevance of the Synthetic Pyrethroid Metofluthrin-Induced Liver Tumors in Rats Based on Mode of Action

In recent years, mode of action (MOA) frameworks have been developed through the International Life Sciences Institute Risk Science Institute and the International Programme on Chemical Safety, including an evaluation of the human relevance of the animal MOA data. In the present paper, the MOA for rat liver tumors induced by Metofluthrin is first analyzed through this framework based on data from studies on Metofluthrin and information on related chemicals from the literature. The human relevance of the rat liver carcinogenic response is then discussed based upon the human relevance framework. Two-year treatment with high dose of Metofluthrin produced hepatocellular tumors in both sexes of the Wistar rats. Metofluthrin induced CYP2B (increased smooth endoplasmic reticulum), resulted in increased liver weights which were associated with centrilobular hepatocyte hypertrophy, and induction of increased hepatocellular DNA replications. The above parameters related to the key events in Metofluthrin-induced liver tumors were observed at or below tumorigenic dose levels. Furthermore, CYP2B induction by Metofluthrin was shown to involve activation of the constitutive androstane receptor in rat hepatocytes. Based on the evidence, including a comparison with the results with another chemical, phenobarbital, acting by a similar MOA, it is reasonable to conclude that Metofluthrin will not have any hepatocarcinogenic activity in humans.

Behavior of α2u-globulin accumulating in kidneys of male rats treated with d-limonene: Kidney-type α2u-globulin in the urine as a marker of d-limonene nephropathy

Abstract Effects of d -limonene on α 2 u -globulin in the kidneys, urine and serum were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis. Treatment of male rats with d -limonene by gavage for 14 consecutive days (300 mg/kg/day) caused accumulation of hyaline droplets in renal proximal tubule cells, and a marked intensification of a protein band corresponding to the kidney-type α 2 u -globulin, with a molecular weight calculated to be approximately 16 kDa. However, no significant changes in the serum α 2 u -globulin (native-type) band, of approximately 19 kDa, were observed between treated rats and controls, suggesting that circulating α 2 u -globulin levels were not affected by the d -limonene administration. While the molecular weight of the major α 2 u -globulin in the urine from control rats was the same as that in the serum (native-type), marked increase in the protein band corresponding to kidney-type- α 2 u -globulin was observed in the urine from treated rats. The results were indicative of elimination of α 2 u -globulin from the kidney to urine, the appearance of kidney-type- α 2 u -globulin in urine implying disruption or exfoliation of proximal tubule cells. Therefore, it is suggested that the presence of the α 2 u -globulin (kidney-type) in the urine might be used as an indicator of chemically induced α 2 u -globulin nephropathy.

α2-Macroglobulin: A Novel Cytochemical Marker Characterizing Preneoplastic and Neoplastic Rat Liver Lesions Negative for Hitherto Established Cytochemical Markers

We tried to identify a novel marker characteristic for rat hepatocellular preneoplastic and neoplastic lesions, undetectable by well established cytochemical markers. Glutathione S-transferase placental (GST-P)-negative hepatocellular altered foci (HAF), hepatocellular adenoma (HCA), and hepatocellular carcinoma (HCC) were generated by two initiation-promotion models with N-nitrosodiethylamine (NDEN) and peroxisome proliferators, Wy-14,643 and clofibrate. Total RNAs isolated from laser-microdissected GST-P-negative HAF (amphophilic cell foci) and adjacent normal tissues were applied to microarray analysis. As a result, five up-regulated genes were identified, and further detailed examinations of the gene demonstrating most fluctuation, ie, that for alpha(2)-macroglobulin (alpha(2)M) were performed. In reverse transcriptase-polymerase chain reaction, alpha(2)M mRNA was overexpressed not only in amphophilic GST-P-negative HAF but also in amphophilic GST-P-negative HCA and HCC. In situ hybridization showed accumulation of alpha(2)M mRNA to be evenly distributed within GST-P-negative HAF (predominantly amphophilic cell foci). Distinctive immunohistochemical staining for alpha(2)M could be consistently demonstrated in GST-P-negative HAF, HCA, and HCC induced not only by peroxisome proliferators but also N-nitrosodiethylamine alone. Thus our findings suggest that alpha(2)M is an important novel cytochemical marker to identify hepatocellular preneoplastic and neoplastic lesions, particularly amphophilic cell foci, undetectable by established cytochemical markers and is tightly linked to rat hepatocarcinogenesis.

α2u-Globulins in the urine of male rats: a reliable indicator for α2u-globulin accumulation in the kidney

Abstract Increases in kidney-type- α 2 u -globulin (αG-K, molecular weight approximately 16 kDa) were detected in the urine of male adult rats treated with d -limonene by immunoblotting analysis using an antiserum which distinguishes native-type- α 2 u -globulin (αG-N, molecular weight approximately 19 kDa) from aG-K. When male adult rats received d -limonene by gavage (0–300 mg/kg/day) for 14 consecutive days, dose-dependent increases in urinary excretion of αG-K were observed at a dosage level of more than 30 mg/kg/day. This was found to be directly correlated with alterations in the concentration of renal αG-K as well as the accumulation of hyaline droplets in proximal convoluted tubule (PCT) epithelial cells in the kidneys. Marked elevation of urinary αG-K was also noted following oral treatment of adult male rats with 2,2,4-trimethylpentane (TMP), 1,4-dichlorobenzene (DCB), decalin and isophorone (ISP) by gavage (1.5 mmol/kg/day) for 7 consecutive days, again in association with increased concentrations of renal αG-K and hyaline droplet accumulation in renal PCT epithelial cells. However, no such increases in urinary αG-K were observed for male adult rats treated with nephrotoxic chemicals such as puromycin aminonucleoside (PAN) (15 mg/kg/day, s.c., 14 consecutive days) or hexachloro-1,3-butadiene (HCBD) (100 mg/kg/day, p.o., 5 consecutive days), lacking the ability to cause kidney accumulation of the hyaline droplets and αG-K. The findings in this study thus indicate that measurement of urinary αG-K can give a reliable estimates not only of the potential to cause renal accumulation of α 2 u -globulin but also of its magnitude.

Spontaneous Iron Accumulation in Hepatocytes of a 7-Week-Old Female Rat

Abstract: Spontaneous iron accumulation in hepatocytes was observed in a 7-week-old female Han Wistar GALAS rat. Very fine yellowish brown pigments, which showed a positive reaction with Berlin Blue stain, were apparent in the cytoplasm close to the bile canaliculi, with a diminishing periportal-to-centrilobular gradient. There were also differences in distribution between and within lobes. Transmission electron microscopy revealed cytosolic ferritin and pericanalicular siderosomes in hepatocytes. No degeneration or necrotic changes were observed, and non-hepatocyte cells did not demonstrate any obvious accumulation of iron. There were no abnormalities in the animal other than this finding in the liver.

Characteristic Upregulation of Glucose-Regulated Protein 78 in an Early Lesion Negative for Hitherto Established Cytochemical Markers in Rat Hepatocarcinogenesis

Previously, we reported α2-macroglobulin (α2M) to be a novel marker characteristic of rat hepatocellular preneoplastic and neoplastic lesions negative for hitherto well-established markers. In the present study, we further examined other candidate markers with specificity for the same type of lesions. Glutathione S-transferase-placental form (GST-P)-negative hepatocellular altered foci (HAF) were generated using a two-stage (initiation and promotion) carcinogenesis protocol with N,N-diethylnitrosamine (DEN) and either Wy-14,643 or clofibrate, two peroxisome proliferators. Microarray analysis using total RNAs isolated from laser-microdissected GST-P-negative HAF (amphophilic cell foci) and adjacent normal tissues was conducted along with immunohistochemistry and real-time RT-PCR. Staining for glucose-regulated protein 78 (GRP78) was detected in GST-P-negative HAF and hepatocellular adenomas, and slightly increased GRP78 mRNA expression was observed in the lesions by real-time RT-PCR analysis. Thus, an early increase of GRP78 expression in hepatocarcinogenesis is likely a feature of the amphophilic subset of HAF.

Ethanol Does Not Promote MeIQx-initiated Rat Colon Carcinogenesis Based on Evidence from Analysis of a Colon Cancer Surrogate Marker

Epidemiological studies suggest that alcohol consumption increases the risk of developing colorectal cancer. However, the data are confounded by numerous cosegregating variables. To cast further light on the relationships between alcohol intake and colon cancer development, 21-day-old male F344/DuCrj rats were fed 200 ppm 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in their diet for 8 weeks and doses of 0, 0.1, 0.3, 1, 3, 10 and 20% of ethanol in their drinking water ad libitum for 16 weeks thereafter. The rats were sacrificed after 24 weeks of experiment, and aberrant crypt foci (ACF), surrogate lesions for colon cancer, were examined under a light microscope at low magnification. Ethanol was found not to affect the ACF formation at any dose compared with the initiated-controls. Furthermore, ethanol did not alter colon epithelial cell proliferation. These data, obtained by analysis of a colon cancer surrogate marker lesion, indicate that ethanol lacks promotion activity for MeIQx-initiated rat colon carcinogenesis.