PBDE-47 and PBDE mixture (DE-71) toxicities and liver transcriptomic changes at PND 22 after in utero/postnatal exposure in the rat.

Pentabromodiphenyl ethers (PBDE) are found in human tissue, in household dust, and in the environment, and a particular concern is the potential for the induction of cancer pathways from these fat-soluble persistent organic pollutants. Only one PBDE cancer study has been conducted and that was for a PBDE mixture (DE-71). Because it is not feasible to test all PBDE congeners in the environment for cancer potential, it is important to develop a set of biological endpoints that can be used in short-term toxicity studies to predict disease outcome after long-term exposures. In this study, PBDE-47 was selected as the test PBDE congener to evaluate and compare toxicity to that of the carcinogenic PBDE mixture. The toxicities of PBDE-47 and the PBDE mixture were evaluated at PND 22 in Wistar Han rat (Crl: WI (Han)) pups after in utero/postnatal exposure (0, 0.1, 15, or 50 mg/kg; dams, GD6-21; pups, PND 12-PND 21; oral gavage daily dosing). By PND 22, PBDE-47 caused centrilobular hypertrophy and fatty change in liver, and reduced serum thyroxin (T4) levels; similar effects were also observed after PBDE mixture exposure. Transcriptomic changes in the liver included induction of cytochrome p450 transcripts and up-regulation of Nrf2 antioxidant pathway transcripts and ABC membrane transport transcripts. Decreases in other transport transcripts (ABCG5 & 8) provided a plausible mechanism for lipid accumulation, characterized by a treatment-related liver fatty change after PBDE-47 and PBDE mixture exposure. The benchmark dose calculation based on liver transcriptomic data was generally lower for PBDE-47 than for the PBDE mixture. The up-regulation of the Nrf2 antioxidant pathway and changes in metabolic transcripts after PBDE-47 and PBDE mixture exposure suggest that PBDE-47, like the PBDE mixture (NTP 2016, TR 589), could be a liver toxin/carcinogen after long-term exposure.

Tetrabromobisphenol A activates the hepatic interferon pathway in rats.

Tetrabromobisphenol A (TBBPA) is a widely used flame retardant in printed circuit boards, paper, and textiles. In a two-year study, TBBPA showed evidence of uterine tumors in female Wistar-Han rats and liver and colon tumors in B6C3F1 mice. In order to gain further insight into early gene and pathway changes leading to cancer, we exposed female Wistar Han rats to TBBPA at 0, 25, 250, or 1000mg/kg (oral gavage in corn oil, 5×/week) for 13 weeks. Because at the end of the TBBPA exposure period, there were no treatment-related effects on body weights, liver or uterus lesions, and liver and uterine organ weights were within 10% of controls, only the high dose animals were analyzed. Analysis of the hepatic and uterine transcriptomes showed TBBPA-induced changes primarily in the liver (1000mg/kg), with 159 transcripts corresponding to 132 genes differentially expressed compared to controls (FDR=0.05). Pathway analysis showed activation of interferon (IFN) and metabolic networks. TBBPA induced few molecular changes in the uterus. Activation of the interferon pathway in the liver occurred after 13-weeks of TBBPA exposure, and with longer term TBBPA exposure this may lead to immunomodulatory changes that contribute to carcinogenic processes.

Characterization of polybrominated diphenyl ether toxicity in Wistar Han rats and use of liver microarray data for predicting disease susceptibilities.

The toxicity of polybrominated diphenyl ethers (PBDEs), flame-retardant components, was characterized in offspring from Wistar Han dams exposed by gavage to a PBDE mixture (DE71) starting at gestation day 6 and continuing to weaning on postnatal day (PND) 21. Offspring from the dams underwent PBDE direct dosing by gavage at the same dose as their dams from PND 12 to PND 21, and then after weaning for another thirteen weeks. Liver samples were collected at PND 22 and week 13 for liver gene expression analysis (Affymetrix Rat Genome 230 2.0 Array). Treatment with PBDE induced 1,066 liver gene transcript changes in females and 1,200 transcriptional changes in males at PND 22 (false discovery rate < 0.01), but only 263 liver transcriptional changes at thirteen weeks in male rats (false discovery rate < 0.05). No significant differences in dose response were found between male and female pups. Transcript changes at PND 22 coded for proteins in xenobiotic, sterol, and lipid metabolism, and cell cycle regulation, and overlapped rodent liver transcript patterns after a high-fat diet or phenobarbital exposure. These findings, along with the observed PBDE-induced liver hypertrophy and vacuolization, suggest that long-term PBDE exposure has the potential to modify cell functions that contribute to metabolic disease and/or cancer susceptibilities.

Activation of peroxisome proliferator-activated receptor gamma (PPARgamma) by rosiglitazone suppresses components of the insulin-like growth factor regulatory system in vitro and in vivo.

Rosiglitazone (Rosi) belongs to the class of thiazolidinediones (TZDs) that are ligands for peroxisome proliferator-activated receptor gamma (PPARgamma). Stimulation of PPARgamma suppresses bone formation and enhances marrow adipogenesis. We hypothesized that activation of PPARgamma down-regulates components of the IGF regulatory system, leading to impaired osteoblast function. Rosi treatment (1 microm) of a marrow stromal cell line (UAMS-33) transfected with empty vector (U-33/c) or with PPARgamma2 (U-33/gamma2) were analyzed by microarray. Rosi reduced IGF-I, IGF-II, IGFBP-4, and the type I and II IGF receptor (IGF1R and IGF2R) expression at 72 h in U-33/gamma2 compared with U-33/c cells (P < 0.01); these findings were confirmed by RT-PCR. Rosi reduced secreted IGF-I from U-33/gamma2 cells by 75% (P < 0.05). Primary marrow stromal cells (MSCs) extracted from adult (8 months) and old (24 months) C57BL/6J (B6) mice were treated with Rosi (1 microm) for 48 h. IGF-I, IGFBP-4, and IGF1R transcripts were reduced in Rosi-treated MSCs compared with vehicle (P < 0.01) and secreted IGF-I was also suppressed (P < 0.05). B6 mice treated with Rosi (20 mg/kg.d) for short duration (i.e. 4 d), and long term (i.e. 7 wk) had reduced serum IGF-I; this was accompanied by markedly suppressed IGF-I transcripts in the liver and peripheral fat of treated animals. To determine whether Rosi affected circulating IGF-I in humans, we measured serum IGF-I, IGFBP-2, and IGFBP-3 at four time points in 50 postmenopausal women randomized to either Rosi (8 mg/d) or placebo. Rosi-treated subjects had significantly lower IGF-I at 8 wk than baseline (-25%, P < 0.05), and at 16 wk their levels were reduced 14% vs. placebo (P = 0.15). We conclude that Rosi suppresses IGF-I expression in bone and liver; these changes could affect skeletal acquisition through endocrine and paracrine pathways.

PPARgamma2 Regulates a Molecular Signature of Marrow Mesenchymal Stem Cells.

Bone formation and hematopoiesis are anatomically juxtaposed and share common regulatory mechanisms. Bone marrow mesenchymal stromal/stem cells (MSC) contain a compartment that provides progeny with bone forming osteoblasts and fat laden adipocytes as well as fibroblasts, chondrocytes, and muscle cells. In addition, marrow MSC provide an environment for support of hematopoiesis, including the development of bone resorbing osteoclasts. The PPARgamma2 nuclear receptor is an adipocyte-specific transcription factor that controls marrow MSC lineage allocation toward adipocytes and osteoblasts. Increased expression of PPARgamma2 with aging correlates with changes in the MSC status in respect to both their intrinsic differentiation potential and production of signaling molecules that contribute to the formation of a specific marrow micro-environment. Here, we investigated the effect of PPARgamma2 on MSC molecular signature in respect to the expression of gene markers associated exclusively with stem cell phenotype, as well as genes involved in the formation of a stem cell supporting marrow environment. We found that PPARgamma2 is a powerful modulator of stem cell-related gene expression. In general, PPARgamma2 affects the expression of genes specific for the maintenance of stem cell phenotype, including LIF, LIF receptor, Kit ligand, SDF-1, Rex-1/Zfp42, and Oct-4. Moreover, the antidiabetic PPARgamma agonist TZD rosiglitazone specifically affects the expression of "stemness" genes, including ABCG2, Egfr, and CD44. Our data indicate that aging and anti-diabetic TZD therapy may affect mesenchymal stem cell phenotype through modulation of PPARgamma2 activity. These observations may have important therapeutic consequences and indicate a need for more detailed studies of PPARgamma2 role in stem cell biology.

The Thermotoga maritima phenotype is impacted by syntrophic interaction with Methanococcus jannaschii in hyperthermophilic coculture.

Significant growth phase-dependent differences were noted in the transcriptome of the hyperthermophilic bacterium Thermotoga maritima when it was cocultured with the hyperthermophilic archaeon Methanococcus jannaschii. For the mid-log-to-early-stationary-phase transition of a T. maritima monoculture, 24 genes (1.3% of the genome) were differentially expressed twofold or more. In contrast, methanogenic coculture gave rise to 292 genes differentially expressed in T. maritima at this level (15.5% of the genome) for the same growth phase transition. Interspecies H2 transfer resulted in three- to fivefold-higher T. maritima cell densities than in the monoculture, with concomitant formation of exopolysaccharide (EPS)-based cell aggregates. Differential expression of specific sigma factors and genes related to the ppGpp-dependent stringent response suggests involvement in the transition into stationary phase and aggregate formation. Cell aggregation was growth phase dependent, such that it was most prominent during mid-log phase and decayed as cells entered stationary phase. The reduction in cell aggregation was coincidental with down-regulation of genes encoding EPS-forming glycosyltranferases and up-regulation of genes encoding beta-specific glycosyl hydrolases; the latter were presumably involved in hydrolysis of beta-linked EPS to release cells from aggregates. Detachment of aggregates may facilitate colonization of new locations in natural environments where T. maritima coexists with other organisms. Taken together, these results demonstrate that syntrophic interactions can impact the transcriptome of heterotrophs in methanogenic coculture, and this factor should be considered in examining the microbial ecology in anaerobic environments.

Functional genomics-based studies of the microbial ecology of hyperthermophilic micro-organisms.

Although much attention has been paid to the genetic, biochemical and physiological aspects of individual hyperthermophiles, how these unique micro-organisms relate to each other and to their natural habitats must be addressed in order to develop a comprehensive understanding of life at high temperatures. Phylogenetic 16 S rRNA-based profiling of samples from various geothermal sites has provided insights into community structure, but this must be complemented with efforts to relate metabolic strategies to biotic and abiotic characteristics in high-temperature habitats. Described here are functional genomics-based approaches, using cDNA microarrays, to gain insight into how ecological features such as biofilm formation, species interaction, and possibly even gene transfer may occur in native environments, as well as to determine what genes or sets of genes may be tied to environmental functionality.