Oxidative stress accelerates intestinal tumorigenesis by enhancing 8-oxoguanine-mediated mutagenesis in MUTYH-deficient mice.

Oxidative stress-induced DNA damage and its repair systems are related to cancer etiology; however, the molecular basis triggering tumorigenesis is not well understood. Here, we aimed to explore the causal relationship between oxidative stress, somatic mutations in pre-tumor-initiated normal tissues, and tumor incidence in the small intestines of MUTYH-proficient and MUTYH-deficient mice. MUTYH is a base excision repair enzyme associated with human colorectal cancer. Mice were administered different concentrations of potassium bromate (KBrO3; an oxidizing agent)-containing water for 4 wk for mutagenesis studies or 16 wk for tumorigenesis studies. All Mutyh -/- mice treated with >0.1% KBrO3 developed multiple tumors, and the average tumor number increased dose dependently. Somatic mutation analysis of Mutyh -/-/rpsL transgenic mice revealed that G:C  > T:A transversion was the only mutation type correlated positively with KBrO3 dose and tumor incidence. These mutations preferentially occurred at 5'G in GG and GAA sequences in rpsL This characteristic mutation pattern was also observed in the genomic region of Mutyh -/- tumors using whole-exome sequencing. It closely corresponded to signature 18 and SBS36, typically caused by 8-oxo-guanine (8-oxoG). 8-oxoG-induced mutations were sequence context dependent, yielding a biased amino acid change leading to missense and stop-gain mutations. These mutations frequently occurred in critical amino acid codons of known cancer drivers, Apc or Ctnnb1, known for activating Wnt signal pathway. Our results indicate that oxidative stress contributes to increased tumor incidence by elevating the likelihood of gaining driver mutations by increasing 8-oxoG-mediated mutagenesis, particularly under MUTYH-deficient conditions.

Characteristic mutations induced in the small intestine of Msh2-knockout gpt delta mice.

BACKGROUND: Base pair mismatches in genomic DNA can result in mutagenesis, and consequently in tumorigenesis. To investigate how mismatch repair deficiency increases mutagenicity under oxidative stress, we examined the type and frequency of mutations arising in the mucosa of the small intestine of mice carrying a reporter gene encoding guanine phosphoribosyltransferase (gpt) and in which the Msh2 gene, which encodes a component of the mismatch repair system, was either intact (Msh2+/+::gpt/0; Msh2-bearing) or homozygously knockout (KO) (Msh2-/-::gpt/0; Msh2-KO). RESULTS: Gpt mutant frequency in the small intestine of Msh2-KO mice was about 10 times that in Msh2-bearing mice. Mutant frequency in the Msh2-KO mice was not further enhanced by administration of potassium bromate, an oxidative stress inducer, in the drinking water at a dose of 1.5 g/L for 28 days. Mutation analysis showed that the characteristic mutation in the small intestine of the Msh2-KO mice was G-to-A transition, irrespective of whether potassium bromate was administered. Furthermore, administration of potassium bromate induced mutations at specific sites in gpt in the Msh2-KO mice: G-to-A transition was frequently induced at two known sites of spontaneous mutation (nucleotides 110 and 115, CpG sites) and at nucleotides 92 and 113 (3'-side of 5'-GpG-3'), and these sites were confirmed to be mutation hotspots in potassium bromate-administered Msh2-KO mice. Administration of potassium bromate also induced characteristic mutations, mainly single-base deletion and insertion of an adenine residue, in sequences of three to five adenine nucleotides (A-runs) in Msh2-KO mice, and elevated the overall proportion of single-base deletions plus insertions in Msh2-KO mice. CONCLUSIONS: Our previous study revealed that administration of potassium bromate enhanced tumorigenesis in the small intestine of Msh2-KO mice and induced G-to-A transition in the Ctnnb1 gene. Based on our present and previous observations, we propose that oxidative stress under conditions of mismatch repair deficiency accelerates the induction of single-adenine deletions at specific sites in oncogenes, which enhances tumorigenesis in a synergistic manner with G-to-A transition in other oncogenes (e.g., Ctnnb1).

Oxidative-stress-driven mutagenesis in the small intestine of the gpt delta mouse induced by oral administration of potassium bromate.

Tumorigenesis induced by oxidative stress is thought to be initiated by mutagenesis, but via an indirect mechanism. The dose-response curves for agents that act by this route usually show a threshold, for unknown reasons. To gain insight into these phenomena, we have analyzed the dose response for mutagenesis induced by the oral administration of potassium bromate, a typical oxidative-stress-generating agent, to gpt delta mice. The agent was given orally for 90 d to either Nrf2+ or Nrf2-knockout (KO) mice and mutants induced in the small intestine were analyzed. In Nrf2+mice, the mutant frequency was significantly greater than in the vehicle controls at a dose of 0.6 g/L but not at 0.2 g/L, indicating that a practical threshold for mutagenesis lies between these doses. At 0.6 g/L, the frequencies of G-to-T transversions (landmark mutations for oxidative stress) and G-to-A transitions were significantly elevated. In Nrf2-KO mice, too, the total mutant frequency was increased only at 0.6 g/L. G-to-T transversions are likely to have driven tumorigenesis in the small intestine. A site-specific G-to-T transversion at guanine (nucleotide 406) in a 5'-TGAA-3' sequence in gpt, and our primer extension reaction showed that formation of the oxidative DNA base modification 8-oxo-deoxyguanosine (8-oxo-dG) at nucleotide 406 was significantly increased at doses of 0.6 and 2 g/L in the gpt delta mice. In the Apc oncogene, guanine residues in the same or similar sequences (TGAA or AGAA) are highly substituted by thymine (G-to-T transversions) in potassium bromate-induced tumors. We propose that formation of 8-oxo-dG in the T(A)GAA sequence is an initiating event in tumor formation in the small intestine in response to oxidative stress.

Mutant Frequency is not Increased in Mice Orally Exposed to Sodium Dichromate.

The in vivo mutagenicity of hexavalent chromium in the small intestine, the target organ of tumorgenicity, was examined by means of a transgenic mouse gene mutation assay. Sodium dichromate dihydrate was administered orally in drinking water to male gpt delta mice at a dose of 85.7 or 257.4 mg/L for 28 days or at a dose of 8.6, 28.6 or 85.7 mg/L for 90 days. No significant increase in gpt mutant frequency relative to that in control mice was observed in the small intestine in either the 28- or 90-day study, whereas 28-day oral administration of potassium bromate, a positive control substance, increased mutant frequency.

Estimation of long-term dietary exposure to acrylamide of the Japanese people.

Acrylamide is a probable human carcinogen and known human neurotoxin. This study estimated hypothetical long-term dietary exposure to acrylamide of the Japanese people using probabilistic and deterministic approaches by combining the concentration of acrylamide in foods with the amount and frequency of food consumption in the population. Data included acrylamide concentrations in more than 2400 individual food samples from a national survey and the literature from 2004 to 2013. Food consumption amounts were derived from the data of 24,293 Japanese citizens aged 1 year and older in the 2012 National Health and Nutrition Survey. Median lifetime average dietary exposure to acrylamide was estimated as 147-154 ng/kg body weight (bw)/day (95th percentile, 226-261 ng/kg bw/day). The deterministic estimate of lifetime exposure was 158 ng/kg bw/day and ranged from 119 ng/kg bw/day for the period of life after 60 years old to 409 ng/kg bw/day for the period between 1 and 6 years old. This study found that vegetables cooked at a high temperature, coffee and cooked potato were the major food groups contributing to long-term dietary acrylamide exposure of the Japanese people.

Evaluation of in vivo mutagenesis for assessing the health risk of air pollutants.

Various kind of chemical substances, including man-made chemical products and unintended products, are emitted to ambient air. Some of these substances have been shown to be mutagenic and therefore to act as a carcinogen in humans. National pollutant inventories (e.g., Pollutant Release and Transfer Registration in Japan) have estimated release amounts of man-made chemical products, but a major concern is the release of suspended particulate matter containing potent mutagens, for example, polycyclic aromatic hydrocarbons and related compounds generated by the combustion of fossil fuel, which are not estimated by PRTR system. In situ exposure studies have revealed that DNA adducts in the lung, and possibly mutations in germline cells are induced in rodents by inhalation of ambient air, indicating that evaluating in vivo mutations is important for assessing environmental health risks. Transgenic rodent systems (Muta, Big Blue, and gpt delta) are good tools for analyzing in vivo mutations induced by a mixture of chemical substances present in the environment. Following inhalation of diesel exhaust (used as a model mixture), mutation frequency was increased in the lung of gpt delta mice and base substitutions were induced at specific guanine residues (mutation hotspots) on the target transgenes. Mutation hotspots induced by diesel exhaust were different from those induced by benzo[a]pyrene, a typical mutagen in ambient air, but nearly identical to those induced by 1,6-dinitropyrene contained in diesel exhaust. Comparison between mutation hotspots in the TP53 (p53) gene in human lung cancer (data extracted from the IARC TP53 database) and mutations we identified in gpt delta mice showed that G to A transitions centered in CGT and CGG trinucleotides were mutation hotspots on both TP53 genes in human lung cancers and gpt genes in transgenic mice that inhaled diesel exhaust. The carcinogenic potency (TD50 value) of genotoxic carcinogen was shown to be correlated with the in vivo mutagenicity (total dose per increased mutant frequency). These results suggest that the mutations identified in transgenic rodents can help identify environmental mutagens that cause cancer.

In vivo mutagenicity of arsenite in the livers of gpt delta transgenic mice.

While arsenic has been classified as a Group 1 human carcinogen by the International Agency for Research on Cancer (IARC), its mutagenicity has not been fully characterized in experimental animals. The aim of this study was to assess the in vivo mutagenicity of arsenite in C57BL/6J gpt delta mice. Male gpt delta mice were given drinking water containing sodium arsenite for 3 weeks, and the hepatic genome was assayed for mutations 2 weeks later. The gpt mutation assays showed a significant increase in mutation frequency in the liver following arsenite exposure. Sequence analysis revealed that 67% of mutations detected are G:C to A:T transitions and 5% are G:C to T:A transversions in the control group, and arsenite exposure resulted in a markedly higher rate of G:C to T:A transversions (46% of mutations detected). G:C to T:A transversions have been reported to be induced following formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a representative product that results from oxidative DNA damage. We also detected a significant increase in 8-OHdG in the livers of the mice exposed to arsenite. These results demonstrate that arsenite has mutagenicity in vivo and suggest that arsenite induces G:C to T:A transversions through oxidative-stress-induced 8-OHdG formation.

Establishment of transactivation assay systems using fish, amphibian, reptilian and human thyroid hormone receptors.

Thyroid hormones are essential for the regulation of a wide range of biological processes associated with normal development and metabolism in vertebrates. For the screening of chemicals with a potential thyroid hormone and anti-thyroid hormone activities, we have established transient transactivation assay systems using thyroid hormone receptors (TRα and TRß) from three frog species (Xenopus laevis, Silurana tropicalis and Rana rugosa), a fish (Oryzias latipes), an alligator (Alligator mississippiensis) and a human (Homo sapiens). In all species examined, similar transcriptional activities were found for triiodothyronine (T3 : 10(-11) M in TRα and 10(-10) M in TRß) and thyroxine (T4 : 10(-9) M in TRα and 10(-8) M in TRß). Analogs of thyroid hormone (3,5,3',-triiodothyroacetic acid and 3,3',5,5'-tetraiodothyroacetic acid) exhibited weaker activity, requiring 10-fold higher concentrations for induction of activity when compared with T3 and T4 . These results provide support for the usefulness of in vitro screening assay systems as part of an approach to test chemicals for potential thyroid hormone receptor activity. In addition, we observed that T3 -stimulated transcriptional activity of the O. latipes TRα was inhibited by 10(-5) M tetrabromobisphenol A (TBBPA). In contrast, TR antagonist activities on TRα were not encountered in other species, even with TBBPA concentrations at 10(-5) M. In vitro transactivation assay systems using TRs from various species can be used for the screening of chemicals with thyroid-receptor agonist and antagonist activities. They also can be used for studies that examine evolutionary differences among species in the potency of TR activation.

Mutations in the lungs of gpt delta transgenic mice following inhalation of diesel exhaust.

Diesel exhaust (DE) is a major airborne pollutant of urban areas. It contains various polycyclic aromatic hydrocarbons (PAH) and nitrated PAHs. In this study, gpt delta mice were treated with inhalation of 1 or 3 mg m(-3) DE, or a single intratracheal instillation of diesel exhaust particles (DEP) or DEP extract. In the lungs of mice treated with inhalation of 3 mg m(-3) DE for 12 weeks, the mutant frequency (MF) was 3.2-fold higher than that of the control group (1.90 x 10(-5) and 0.59 x 10(-5), respectively). An instillation of DEP and DEP extract resulted in a significant dose-dependent linear increase in MF. In mice treated with 0.5 mg DEP and 0.2 mg DEP extract, the MFs were 3.0- and 2.7-fold higher than that of the control group, respectively. The mutagenic potency (MF mg(-1)) of DEP extract (5.6 x 10(-5)) was double that of DEP (2.7 x 10(-5)), suggesting that the mutagenicity of the latter is derived primarily from compounds in the extract, which itself is responsible for ca. 50% of the weight of DEP. G:C-->A:T transitions were the predominant gpt mutation induced by all three treatments and G:C-->T:A transversions were induced by DEP and DEP extract. Guanine bases centered in nucleotide sequences such as GGA, TGA, CGG, and CGT were the major mutation targets of all three treatments. Thus, our results suggest that the mutagens contained in DEP such as PAH and nitrated PAHs induce mutations and may be responsible for carcinogenesis caused by inhalation of DE.

Enhanced spontaneous and benzo(a)pyrene-induced mutations in the lung of Nrf2-deficient gpt delta mice.

The lung is an organ that is sensitive to mutations induced by chemicals in ambient air, and transgenic mice harboring guanine phosphoribosyltransferase (gpt) gene as a target gene are a well-established model system for assessing genotoxicity in vivo. Transcription factor Nrf2 mediates inducible and constitutive expression of cytoprotective enzymes against xenobiotics and mutagens. To address whether Nrf2 is also involved in DNA protection, we generated nrf2+/-::gpt and nrf2-/-::gpt mice. The spontaneous mutation frequency of the gpt gene in the lung was approximately three times higher in nrf2-null (nrf2-/-) mice than nrf2 heterozygous (nrf2+/-) and wild-type (nrf2+/+) mice, whereas in the liver, the mutation frequency was higher in nrf2-/- and nrf2+/- mice than in nrf2+/+ wild-type mice. By contrast, no difference in mutation frequency was observed in testis among the three genotypes. A single intratracheal instillation of benzo(a)pyrene (BaP) increased the lung mutation frequency 3.1- and 6.1-fold in nrf2+/- and nrf2-/- mice, respectively, compared with BaP-untreated nrf2+/- mice, showing that nrf2-/- mice are more susceptible to genotoxic carcinogens. Surprisingly, mutation profiles of the gpt gene in BaP-treated nrf2+/- mice was substantially different from that in BaP-untreated nrf2-/- mice. In nrf2-/- mice, spontaneous and BaP-induced mutation hotspots were observed at nucleotides 64 and 140 of gpt, respectively. These results thus show that Nrf2 aids in the prevention of mutations in vivo and suggest that Nrf2 protects genomic DNA against certain types of mutations.

Increased susceptibility of MER5 (peroxiredoxin III) knockout mice to LPS-induced oxidative stress.

MER5 (also called peroxiredoxin III, PrxIII) is a member of peroxiredoxin family that has antioxidant activity. The present study was performed to investigate its in vivo function using MER5 knockout mice. MER5 knockout mice were born in normal frequency and could grow to maturity, but we found that intracellular ROS levels are significantly higher in the macrophages of the knockout mice. We examined roles of MER5 function for the oxidative stress responses by intratracheal inoculation of lipopolysaccharide (LPS) to the mice. Lung inflammation such as inflammatory cell infiltration and airway wall thickening was more severely detected in the knockout mice. At the same time, oxidative damage on DNA and proteins was more strongly detected in lung tissues of the knockout mice, including 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation and protein carbonylation. The degrees of lung inflammation and oxidative damage were positively related with LPS doses. Our results indicate that MER5 knockout mice accumulated higher intracellular ROS levels, which cause LPS-induced lung injury more severely, and thus, suggested that MER5 acts as an important scavenger of reactive oxygen species (ROS) under oxidative stress.

Quantitative risk analysis of particulate matter in the air: interspecies extrapolation with bioassay and mathematical models.

We analyzed the health risk of particulate matters in the air to humans using bioassay data and a mathematical model. We designed an original dosimetry model to estimate the particle concentration in human respiratory organs, and the concentration of the inhaled particles at the target organ was used for interspecies extrapolation from rat to human. Our model is based on the conventional dosimetry model and deposition model in the previous literature, but clearance parameters have been newly introduced for the simulation of long-term exposure. Lung cancer was set as the risk endpoint in our risk study, and the dose-response relationship at the target organ (lung) was quantitatively analyzed by the benchmark dose (BMD) method. For interspecies extrapolation based on target organ concentration, we assumed benchmark concentration (BMC) related to 1% excess cancer in rats and humans, and the human equivalent concentration (HEC) was searched by back-estimation using our model. The obtained HEC was 948 to 1098 mg/m3, and the unit risk to humans was 9.11 to 10.5 x 10(-9) per 1 microg/m3 of particulate matter. The estimated cancer risk for Japanese people in general was estimated as approximately 9-10 persons per 100,000,000 when the particle concentration in the air is 10 microg/m3.

Estimated cancer risk of dioxins to humans using a bioassay and physiologically based pharmacokinetic model.

The health risk of dioxins and dioxin-like compounds to humans was analyzed quantitatively using experimental data and mathematical models. To quantify the toxicity of a mixture of three dioxin congeners, we calculated the new relative potencies (REPs) for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), and 2,3,4,7,8- pentachlorodibenzofuran (PeCDF), focusing on their tumor promotion activity. We applied a liver foci formation assay to female SD rats after repeated oral administration of dioxins. The REP of dioxin for a rat was determined using dioxin concentration and the number of the foci in rat liver. A physiologically based pharmacokinetic model (PBPK model) was used for interspecies extrapolation targeting on dioxin concentration in liver. Toxic dose for human was determined by back-estimation with a human PBPK model, assuming that the same concentration in the target tissue may cause the same level of effect in rats and humans, and the REP for human was determined by the toxic dose obtained. The calculated REPs for TCDD, PeCDD, and PeCDF were 1.0, 0.34, and 0.05 for rats, respectively, and the REPs for humans were almost the same as those for rats. These values were different from the toxic equivalency factors (TEFs) presented previously (Van den Berg, M., Birnbaum, L., Bosveld, A.T.C., Brunstrom, B., Cook, P., Feeley, M., Giesy, J.P., Hanberg, A., Hasegawa, R., Kennedy, S.W., Kubiak, T., Larsen, J.C., Rolaf van Leeuwen, F.X., Liem, A.K.D., Nolt, C., Peterson, R.E., Poellinger. L., Safe, S., Schrenk, D., Tillitt, D, Tysklind, M., Younes, M., Waern, F., Zacharewski, T., 1998. Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife. Environ. Health Perspect. 106, 775-792). The relative risk of excess liver cancer for Japanese people in general was 1.7-6.5 x 10(-7) by TCDD only, and 2.9-11 x 10(-7) by the three dioxins at the present level of contamination.

An essential role for the His-8 residue of the SDF-1alpha-chimeric, tropism-redirected Env protein of the Moloney murine leukemia virus in regulating postbinding fusion events.

BACKGROUND: To use retroviral vectors for the cell-specific delivery of genes, it is necessary to redirect their receptor tropism to cell-specific receptors. Previously, we reported that a Moloney murine leukemia virus (MLV) retroviral vector containing a human stromal-derived factor-1alpha (SDF-1alpha)-chimeric envelope protein (Env) (S3) acquired the ability to transduce human cells via CXCR4, the cognate receptor for SDF-1alpha, while retaining the ability to transduce mouse cells via mCAT1. METHODS: We constructed expression plasmids for derivatives of the S3 Env protein; S3-D84K containing an Asp-84-to-Lys (D84K) substitution, S3-H8R-D84K containing D84K and an additional His-8-to-Arg substitution, and S3-D84K-RY containing D84K and additional Gln-227-to-Arg plus Asp-243-to-Tyr substitutions which have been suggested to suppress the loss of function of His-8. Cellular expression, virion incorporation, and entry functions of these derivatives were investigated. RESULTS: All three derivatives were incorporated into virions. The S3-D84K vector lost its ecotropism, but could transduce CXCR4-expressing human and mouse cells at titers of 10(3) to 10(4) colony-forming units (cfu)/ml. The S3-H8R-D84K vector did not show transduction, although its Env protein could bind to CXCR4. The transduction titer of the S3-D84K-RY vector via CXCR4 was slightly lower than that of the S3-D84K vector. These results indicate that the His-8 residue of the S3-D84K Env protein is indispensable and may be fully functional in postbinding membrane fusion. CONCLUSIONS: Insertion of a ligand at Pro-79 of the Moloney MLV Env protein has proved to be a valuable strategy for constructing direct targeting retroviral vectors, since it permits the formation of a redirected Env protein without ecotropism, and it does not disrupt the function of the essential His-8 residue.

Dioxin health risk to infants using simulated tissue concentrations.

Dioxin concentrations in infant and child were simulated using physiologically based pharmacokinetic (PBPK) models developed for these groups. The infant model was validated by comparing the simulated concentration with the measured concentration from the literature, and they showed good agreement. Simulations with our PBPK model showed temporal patterns in concentrations in various tissues. For risk assessment, estimated concentrations of 29 dioxins in the liver were summed up in a toxic equivalency (TEQ) basis to be compared with actual 2,3,7,8-TCDD concentrations in rat liver associated with toxicity. Maximum liver concentrations in breast-fed and formula-fed infants were 16.8pg TEQ/g and 3.5pg TEQ/g, respectively. The level in breast-fed infant liver was approximately 1/300 of the level associated with hepatocellular carcinoma and 1/5 of the level found in maternal rat liver associated with alterations in reproductive organs in the next generation. Based on our analysis, the present contamination level is not safe enough, but further dose-response data is required for a quantitative risk assessment.

Altered protein profile and possible hypoxia in the placenta of 2,3,7,8-tetrachlorodibenzo-p-dioxin-exposed rats.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during pregnancy causes fetal death in many animal species. In an earlier study we observed alteration of placental glucose kinetics at the same TCDD exposure level that resulted in fetal death (Ishimura et al., Toxicol. Appl. Pharmacol. 178, 161-171, 2002). In the present study, in order to identify the molecules that might explain the alterations of placental function and the mechanism of fetal death, we used two-dimensional gel electrophoresis (2D/E) to detect and identify placental proteins whose amounts changed after exposure to TCDD and we examined the expression properties of these proteins in the placenta during hypoxia by using the uterine artery ligation model. Pregnant Holtzman rats were given a single oral dose of 1600 ng TCDD/kg body wt or an equivalent volume of vehicle (control) on gestational day (GD) 15 and placental tissue was collected on GD16 and GD20. The 15,000 g supernatant fractions of placental homogenates from the control group and TCDD-exposed group were subjected to the 2D/E analysis, and the protein spots whose amounts had changed after exposure to TCDD were characterized by amino acid sequence analysis. The amounts of heat shock protein 27 (Hsp27) and beta-tropomyosin (beta-TM) in TCDD-exposed placentas tended to have increased on GD16 and had increased significantly on GD20, and these changes were followed by an approximately twofold increase in glyceraldehyde 3-phosphate dehydrogenase (GAPDH) on GD20. Next, the uterine-artery ligation model was prepared on GD15, and the hypoxic placentas were collected on GD20. Two-D/E analysis of the 15,000 g supernatant proteins of the placentas revealed an increased level of GAPDH but not of other proteins, including Hsp27 and beta-TM. The results of this study showed that the increase in GAPDH level during hypoxia previously observed in endothelial cells occurs in the placenta and indicated that the TCDD-exposed placentas were in a hypoxic state at the end of pregnancy. Finally, the results of this study suggested the possibility that the increased incidence of fetal death after exposure to TCDD was due to the placental hypoxia.

Paradoxical increase of heat-shock response with age in a substrain of F344 rats: comparison between F344/DuCrj and F344/Jcl.

The ability of hepatocytes isolated from young (7-10 months) and old (31 months) male F344/Jcl and F344/DuCrj rats to express heat shock protein (hsp) 27, hsp70 and hsp90 was determined after a mild heat shock (42.5 degrees C for 30 min). The induction of these three mRNA levels by the heat shock was 50-80% lower in hepatocytes isolated from old F344/Jcl rats than in those from young rats. However, the hepatocytes from old F344/DuCrj showed a marked increase (200-250%) in the induction of hsp mRNAs by heat shock when compared to cells from young rats. Because heat shock transcription factor (HSF) plays a critical role in regulating the transcription of hsp genes, the effect of age on the binding activity HSF to heat shock element (HSE) was also studied. Again, the induction of binding activity of HSF to HSE was significantly increased with age in hepatocytes from F344/DuCrj rats while the reverse was true for the cells from F344/Jcl. The induced levels of hsp mRNAs were positively correlated with the binding activity of HSF to HSE in hepatocyte extracts from both F344 substrains, suggesting that the diverse age-related changes of heat-shock response in F344 substrains occurs in HSF activity. The contradictory age-related change in the heat-shock response is discussed with the differences in biochemical and genetic properties of substrains of F344 rats.

Increased glycogen content and glucose transporter 3 mRNA level in the placenta of Holtzman rats after exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Exposure to a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces a variety of toxic manifestations, including fetal death. In order to evaluate the effects of low-dose TCDD on placental function in this study, pregnant Holtzman rats were given a single oral dose of 800 or 1600 ng TCDD/kg body wt or an equivalent volume of vehicle (control) on gestation day (GD) 15 and the results were observed on GD16 and GD20. The number of fetal deaths increased in the animals exposed to TCDD. Although fetal and placental weight did not differ significantly between the control group and the TCDD groups, histological differences from the control rats were clearly observed in the junctional zone (JZ) of the placentas of the TCDD-exposed rats. In the control placenta, glycogen cells occupied the majority of the JZ on GD16, but then decreased in number and almost disappeared by GD20, whereas on GD20 the placenta of the TCDD-exposed rats exhibited a larger area occupied by the glycogen cells and cysts filled with eosinophilic material surrounded by glycogen cells in the JZ than that of the control group. Glycogen assay revealed that the glycogen content of the placentas from the TCDD-exposed rats was higher than in the control rats. Semiquantitative RT-PCR analysis was performed to assess the expression of glucose transporter 1 (GLUT1) and GLUT3, the two major placental glucose transporter isoforms. On GD20 the level of expression of GLUT1 mRNA in the placentas was not different between the control and TCDD groups, whereas the level of expression of GLUT3 mRNA approximately doubled in both the 800 and 1600 ng/kg TCDD groups. GLUT3 mRNA expression was restricted to the labyrinth zone of placenta, where zone-specific expression of mRNA arylhydrocarbon receptor and induction of cytochrome P450 1A1 mRNA by TCDD were observed, and none was detected in the JZ. These results, including the increase of glycogen content and GLUT3 mRNA level in TCDD-exposed placentas, provide the first evidence of alteration of glucose kinetics in the placenta by TCDD.