F344/NTac Rats Chronically Exposed to Bromodichloroacetic Acid Develop Mammary Adenocarcinomas With Mixed Luminal/Basal Phenotype and Tgfß Dysregulation.

Breast cancer is the most common cancer and the second-leading cause of cancer mortality in women in the United States. A recent 2-year National Toxicology Program carcinogenicity study showed an increased incidence of proliferative mammary lesions (hyperplasia, fibroadenoma, adenocarcinoma) in F344/NTac rats exposed to bromodichloroacetic acid (BDCA), a disinfection by-product in finished drinking water with widespread human exposure. We hypothesized that the increase in mammary tumors observed in BDCA-exposed F344/NTac rats may be due to underlying molecular changes relevant for human breast cancer. The objective of the study was to compare (1) gene and protein expression and (2) mutation spectra of relevant human breast cancer genes between normal untreated mammary gland and mammary tumors from control and BDCA-exposed animals to identify molecular changes relevant for human cancer. Histologically, adenocarcinomas from control and BDCA-exposed animals were morphologically very similar, were estrogen/progesterone receptor positive, and displayed a mixed luminal/basal phenotype. Gene expression analysis showed a positive trend in the number of genes associated with human breast cancer, with proportionally more genes represented in the BDCA-treated tumor group. Additionally, a 5-gene signature representing possible Tgfß pathway activation in BDCA-treated adenocarcinomas was observed, suggesting that this pathway may be involved in the increased incidence of mammary tumors in BDCA-exposed animals.

H-ras consensus sequence and mutations in primary hepatocellular carcinomas of lemurs and lorises.

The authors have determined a consensus sequence for exons 1 and 2 of H-ras from captive lemurs and lorises and evaluated samples of nonneoplastic liver and hepatocellular carcinomas (HCC) from affected animals for mutations in these exons. Frozen liver samples were collected from 20 animals representing 9 different species with a sex distribution of 10 males and 10 females. A total of 26 liver samples, including 11 normal livers, 9 HCC, and 6 samples from nonneoplastic regions of liver from animals with HCC, were evaluated. This is the first report of the consensus sequence for exons 1 and 2 of H-ras in prosimians, and the authors have determined that it is identical to that of human H-ras and differs only slightly from the chimpanzee sequence. Point mutations were identified in 6 of the 9 HCC samples examined with codons 7, 22, 32, 56, 61, 84, and 96 affected. Two carcinomas had double mutations, and one tumor had triple mutations. One HCC had a mutation in codon 61, which is identical to a recognized affected codon for an H-ras "hot spot" in rodent neoplasia that has also been reported in human tumors. Although not statistically different, metastasis occurred in 5 of 6 HCC with H-ras mutation and only 1 of 3 HCC without mutations. There were 4 silent mutations that did not contain changes in the encoded amino acids, 2 of which were found in nonneoplastic regions of tumor-bearing liver.

Gene expression and mutation assessment provide clues of genetic and epigenetic mechanisms in liver tumors of oxazepam-exposed mice.

Liver tumors from a previous National Toxicology Program study were examined using global gene expression and mutation analysis to define the mechanisms of carcinogenesis in mice exposed to oxazepam. Five hepatocellular adenomas and 5 hepatocellular carcinomas from male B6C3F1 mice exposed to 5000 ppm oxazepam and 6 histologically normal liver samples from control animals were examined. One of the major findings in the study was upregulation of the Wnt/ß-catenin signaling pathway. Genes that activate ß-catenin, such as Sox4, were upregulated, whereas genes that inhibit Wnt signaling, such as APC and Crebbp, were downregulated. In addition, liver tumors from oxazepam-exposed mice displayed ß-catenin mutations and increased protein expression of glutamine synthetase, a downstream target in the Wnt signaling pathway. Another important finding in this study was the altered expression of oxidative stress-related genes, specifically increased expression of cytochrome p450 genes, including Cyp1a2 and Cyp2b10, and decreased expression of genes that protect against oxidative stress, such as Sod2 and Cat. Increased oxidative stress was confirmed by measuring isoprostane expression using mass spectrometry. Furthermore, global gene expression identified altered expression of genes that are associated with epigenetic mechanisms of cancer. There was decreased expression of genes that are hypermethylated in human liver cancer, including tumor suppressors APC and Pten. Oxazepam-induced tumors also exhibited decreased expression of genes involved in DNA methylation (Crebbp, Dnmt3b) and histone modification (Sirt1). These data suggest that formation of hepatocellular adenomas and carcinomas in oxazepam-exposed mice involves alteration of the Wnt signaling pathway, oxidative stress, and potential epigenetic alterations.

Respiratory sensation during chest wall restriction and dead space loading in exercising men.

We mimicked important mechanical and ventilatory aspects of restrictive lung disorders by employing chest wall strapping (CWS) and dead space loading (DS) in normal subjects to gain mechanistic insights into dyspnea causation and exercise limitation. We hypothesized that thoracic restriction with increased ventilatory stimulation would evoke exertional dyspnea that was similar in nature to that experienced in such disorders. Twelve healthy young men [28 +/- 2 (SE) yr of age] completed pulmonary function tests and maximal cycle exercise tests under four conditions, in randomized order: 1) control, 2) CWS to 60% of vital capacity, 3) added DS of 600 ml, and 4) CWS + DS. Measurements during exercise included cardiorespiratory parameters, esophageal pressure, and Borg scale ratings of dyspnea. Compared with control, CWS significantly reduced the tidal volume response to exercise, increased dyspnea intensity at any given work rate or ventilation, and thus limited exercise performance. DS stimulated ventilation but had minimal effects on dyspnea and exercise performance. Adding DS to CWS further increased dyspnea by 1.7 +/- 0.6 standardized Borg units (P = 0.012) and decreased exercise performance (total work) by 21 +/- 6% (P = 0.003) over CWS alone. Across conditions, increased dyspnea intensity correlated best with decreased resting inspiratory reserve volume (r = -0.63, P < 0.0005). Dyspnea during CWS was described primarily as "inspiratory difficulty" and "unsatisfied inspiration," similar to restrictive disorders. In conclusion, severe dyspnea and exercise intolerance were provoked in healthy normal subjects when tidal volume responses were constrained in the face of increased ventilatory drive during exercise.

Residual hematopoietic effect in mice exposed to ochratoxin A prior to irradiation.

Ochratoxin A (OCT A) has the potential to cause myelotoxicity in addition to the well-known toxic effects on the liver and kidney. Whereas in previous studies the bone marrow parameters were examined shortly after injection, experiments reported here were designed to determine whether mice would recover from the myelotoxic effects induced to OCT A injection and secondly whether mice previously injected to OCT A would be more sensitive to radiation-induced myelotoxicity than vehicle controls. Six-week old female B6C3F1 mice were injected intraperitoneally on alternate days over a week with a total dose of 20 or 40 mg/kg of OCT A and bone marrow parameters monitored for up to 16 weeks. Controls received vehicle alone. There was a suppression of marrow granulocyte macrophage progenitors (CFU-C) in OCT A-treated animals which returned to normal values by 2 weeks (20 mg/kg group) or by 5 weeks (40 mg/kg group) following the last treatment. Some of the OCT A-treated mice were additionally irradiated with 200 rads of whole body irradiation (WBI) at 10 and 52 days following OCT A injections. Irradiation caused a significant reduction in CFU-Cs in all mice but the effects were more pronounced in the mice that had received OCT A previously. The bone marrow parameters of 40 mg/kg OCT A-treated mice returned to normal by 6 weeks after first WBI. A second WBI produced similar depression in CFU-Cs with again a delayed 8 weeks recovery as compared to controls in both dose groups of OCT A-treated mice. The delayed recovery in bone marrow progenitors was also reflected in lower peripheral white blood counts after the second irradiation in 40 mg/kg OCT A-treated mice as compared to the untreated irradiated controls. This indicated that residual bone marrow effect of OCT A makes the mice more sensitive to subsequent irradiation induced injury.

Connective tissue growth factor in drug-induced gingival overgrowth.

BACKGROUND: Drug-induced gingival overgrowth is a known side effect of certain chemotherapeutic agents used for the treatment of systemic disorders. The pathogenesis and mechanisms responsible for this condition are not fully understood. This study assesses for the presence and localization of connective tissue growth factor (CTGF) in drug-induced gingival overgrowth tissues. CTGF immunostaining was compared with sections stained with transforming growth factor (TGF)-beta1 and CD31 antibodies in order to investigate possible pathogenic mechanisms. METHODS: Gingival overgrowth samples were obtained from patients undergoing therapy with phenytoin (n = 9), nifedipine (n = 4), cyclosporin A (n = 5), and control tissues from systemically healthy donors (n = 9). Tissue sections were subjected to peroxidase immunohistochemistry and were stained with CTGF and TGF-beta1 polyclonal primary antibodies. Possible relationships between CTGF staining and angiogenesis were also studied using an anti-CD31 antibody as a marker for endothelial cells. Staining was analyzed by computer-assisted quantitative and semiquantitative methodology at 5 defined sites in all samples based on the location of specific landmarks including epithelium and underlying connective tissues. RESULTS: Cellular and extracellular CTGF content in phenytoin gingival overgrowth tissues was significantly (P<0.05) higher compared to the other gingival overgrowth tissues and the controls. Higher CTGF staining in phenytoin gingival overgrowth tissues was accompanied by an increased abundance of fibroblasts and connective tissue fibers. No strong association of CTGF staining with TGF-beta1 or CD31 staining was found. CONCLUSIONS: The data from the present study show significantly higher CTGF staining in phenytoin-induced gingival overgrowth tissues compared to controls, cyclosporin A-, or nifedipine-induced gingival overgrowth. Moreover, semiquantitative analyses of histologic samples support the concept that the phenytoin overgrowth tissues are fibrotic. These associations suggest a possible role for CTGF in promoting development of fibrotic lesions in phenytoin-induced gingival overgrowth.

Preserving pulpal health of a geminated maxillary lateral incisor through multidisciplinary care.

AIM: To report the multidisciplinary care of an unaesthetic geminated maxillary lateral incisor tooth, which allowed its preservation in the mouth. SUMMARY: Preoperative examination of an unsightly geminated maxillary lateral incisor (tooth 22) demonstrated two pulp chambers and open apices, with normal pulp sensitivity responses. At surgery, a periodontal mucoperiosteal flap was reflected and the distal part of the geminated tooth was removed. The exposed root canal of the preserved tooth was sealed with mineral trioxide aggregate (MTA). The extraction socket and osseous defect was grafted with decalcified freeze-dried bone allograft (DFDBA) before flap closure. During follow-up, distal caries in tooth 22 and a diastema between tooth 22 and 23 were managed with composite resin restorations. Forty-two months postoperatively, normal thermal and electrical pulp sensitivity tests confirmed pulp health. Convincing apexogenesis and dentinogenesis of the developing root was confirmed by radiographic examination. Acceptable periodontal health including 3-4 mm clinical probing depths was achieved. Optimizing aesthetics and occlusion was accomplished without orthodontic treatment.

Regulation of lysyl oxidase, collagen, and connective tissue growth factor by TGF-beta1 and detection in human gingiva.

Gingival overgrowth is characterized by excess extracellular matrix accumulation and elevated levels of cytokines, including transforming growth factor-beta1 (TGF-beta1). The functional relationships between altered cytokine levels and extracellular matrix accumulation have not been extensively investigated in gingival cells and tissues. Lysyl oxidase catalyzes the final known enzymatic step required for cross-linking collagen and elastin in the synthesis of a functional extracellular matrix. This study investigated the regulation by TGF-beta1 of lysyl oxidase and its collagen and elastin substrates in early passage human gingival fibroblasts. In addition, TGF-beta1 regulation of connective tissue growth factor (CTGF) was assessed in human gingival cells and tissues. The results show that TGF-beta1 increases lysyl oxidase enzyme activity and mRNA levels for lysyl oxidase and alpha-1-type I collagen, but not elastin, in a dose- and time-dependent manner. Maximal stimulation of lysyl oxidase activity and mRNA levels for both lysyl oxidase and collagen occurs after 48 hours of treatment of gingival fibroblastic cells with 400 pM of TGF-beta1. This study shows for the first time that CTGF mRNA and protein are strongly and rapidly induced by TGF-beta1 in human gingival fibroblasts. Exogenous addition of 1 to 50 ng/ml CTGF to gingival fibroblasts stimulates production of lysyl oxidase enzyme activity up to 1.5-fold after 48 hours, and 50 ng/ml CTGF stimulated insoluble collagen accumulation 1.5- to 2.0-fold after 4, 11, and 18 days of treatment. It is interesting to note that the addition of CTGF-blocking antibodies in the presence of TGF-beta did not block TGF-beta stimulation of collagen mRNA levels. Thus, although CTGF itself contributes to increased insoluble collagenous extracellular matrix accumulation, CTGF does not mediate all of the effects of TGF-beta1 on stimulation of collagen mRNA levels in human gingival fibroblasts. Immunohistochemistry studies of gingival overgrowth tissue samples indicate for the first time detectable levels of CTGF protein in Dilantin-induced hyperplasia tissues also positive for TGF-beta1. CTGF was not found in TGF-beta1-negative samples. In addition, extracellular lysyl oxidase protein was detected in vivo. Taken together, these studies support mostly independent roles for TGF-beta1 and CTGF in stimulating collagenous extracellular matrix accumulation in human gingival fibroblasts and tissues.

Mutations of ras protooncogenes and p53 tumor suppressor gene in cardiac hemangiosarcomas from B6C3F1 mice exposed to 1,3-butadiene for 2 years.

1,3-Butadiene is a multisite carcinogen in rodents. Incidences of cardiac hemangiosarcomas were significantly increased in male and female B6C3F1 mice that inhaled 1,3-butadiene (BD) for 2 years. Eleven BD-induced cardiac hemangiosarcomas were examined for genetic alterations in ras protooncogenes and in the p53 tumor suppressor gene. Nine of 11 (82%) BD-induced hemangiosarcomas had K-ras mutations and 5 of 11 (46%) had H-ras mutations. All of the K-ras mutations were G-->C transversions (GGC-->CGC) at codon 13; this pattern is consistent with reported results in BD-induced lung neoplasms and lymphomas. Both K-ras codon 13 CGC mutations and H-ras codon 61 CGA mutations were detected in 5 of 9 (56%) hemangiosarcomas. The 11 hemangiosarcomas stained positive for p53 protein by immunohistochemistry and were analyzed for p53 mutations using cycle sequencing of polymerase chain reaction (PCR) amplified DNA isolated from paraffin-embedded sections. Mutations in exons 5 to 8 of the p53 gene were identified in 5 of 11 (46%) hemangiosarcomas, and all of these were from the 200- or 625-ppm exposure groups that also had K-ras codon 13 CGC mutations. Our data indicate that K-ras, H-ras, and p53 mutations in these hemangiosarcomas most likely occurred as a result of the genotoxic effects of BD and that these mutations may play a role in the pathogenesis of BD-induced cardiac hemangiosarcomas in the B6C3F1 mouse.

Frequency of ras mutations in liver neoplasms from B6C3F1 mice exposed to tetrafluoroethylene for two years.

Tetrafluoroethylene (TFE) was evaluated for carcinogenicity in inhalation studies because of its high use in the production of Teflon. There was clear evidence of hepatocarcinogenic activity in B6C3F1 mice after 2 yr of TFE exposure. The present study was designed to characterize the mutation profiles of H- and K-ras oncogenes in liver neoplasms in mice after exposure to 0, 312, 625, or 1,250 ppm TFE. ras mutations were identified by restriction fragment length polymorphism, single-stranded conformation polymorphism analysis, and direct sequencing of polymerase chain reaction amplified-DNA isolated from frozen or paraffin-embedded liver neoplasms. A low frequency (15%, 9/59) of H-ras codon 61 mutations was detected in hepatocellular neoplasms when compared with the higher frequency (59% of this study and 56% of historical data) in spontaneously occurring liver neoplasms. There was no difference in the mutation frequency or spectrum among exposure groups or between benign and malignant hepatocellular neoplasms. K-ras mutations at codons 12, 13, and 61 and H-ras mutations at codon 117 were not detected in hepatocellular neoplasms. These data suggest that TFE-induced hepatocellular neoplasms are developed by pathways that are mostly independent of ras mutations. The ras mutation frequency and spectrum were similar to those of the structurally related chemical tetrachloroethylene.

Predominant p53 G-->A transition mutation and enhanced cell proliferation in uterine sarcomas of CBA mice treated with 1,2-dimethylhydrazine.

Mouse uterine tumors were examined for genetic alterations in the ras proto-oncogene and p53 tumor suppressor gene and for other biologically relevant immunohistochemical markers that may increase our understanding of the events that occur in uterine cancer. Fourteen dimethylhydrazine (DMH)-induced uterine sarcomas, including 3 primary malignant fibrous histiocytomas (MFH), 7 transplanted MFH, 3 stromal sarcomas, and 1 undifferentiated sarcoma, were first screened by immunohistochemistry for p53 missense mutations, followed by single strand conformation polymorphism analysis and DNA sequencing for the identification of point mutations. There was 100% correlation between p53 protein immunopositivity and subsequent detection of p53 mutations in DMH-induced malignant fibrous histiocytomas. All MFH had a characteristic p53 G:C-->A:T transition mutation, consistent with O6-methylguanine mispairing with thymine, the most common DNA lesion caused by alkylating agents. DMH-induced uterine MFH with p53 mutations also had a higher proliferative rate (qualitatively evaluated by immunohistochemical detection of proliferating cell nuclear antigen) when compared with other DMH-induced sarcomas. Uterine sarcomas were further evaluated for biological end points, such as estrogen receptor and desmin. Neoplastic cells from stromal sarcomas (SS), undifferentiated sarcomas (US), and MFH did not stain for desmin. The estrogen receptor was detected in normal uteri and a small portion of MFH, SS, and US. Our data suggest that DMH-induced uterine sarcomas are not consistent with smooth muscle cell origin and that a subset of these tumors, specifically DMH-induced malignant fibrous histiocytomas, have unique p53 G:C-->A:T transitions and a high proliferative rate.

Multiple bone morphogenetic protein 1-related mammalian metalloproteinases process pro-lysyl oxidase at the correct physiological site and control lysyl oxidase activation in mouse embryo fibroblast cultures.

Lysyl oxidase catalyzes the final enzymatic step required for collagen and elastin cross-linking in extracellular matrix biosynthesis. Pro-lysyl oxidase is processed by procollagen C-proteinase activity, which also removes the C-propeptides of procollagens I-III. The Bmp1 gene encodes two procollagen C-proteinases: bone morphogenetic protein 1 (BMP-1) and mammalian Tolloid (mTLD). Mammalian Tolloid-like (mTLL)-1 and -2 are two genetically distinct BMP-1-related proteinases, and mTLL-1 has been shown to have procollagen C-proteinase activity. The present study is the first to directly compare pro-lysyl oxidase processing by these four related proteinases. In vitro assays with purified recombinant enzymes show that all four proteinases productively cleave pro-lysyl oxidase at the correct physiological site but that BMP-1 is 3-, 15-, and 20-fold more efficient than mTLL-1, mTLL-2, and mTLD, respectively. To more directly assess the roles of BMP-1 and mTLL-1 in lysyl oxidase activation by connective tissue cells, fibroblasts cultured from Bmp1-null, Tll1-null, and Bmp1/Tll1 double null mouse embryos, thus lacking BMP-1/mTLD, mTLL-1, or all three enzymes, respectively, were assayed for lysyl oxidase enzyme activity and for accumulation of pro-lysyl oxidase and mature approximately 30-kDa lysyl oxidase. Wild type cells or cells singly null for Bmp1 or Tll1 all produced both pro-lysyl oxidase and processed lysyl oxidase at similar levels, indicating apparently normal levels of processing, consistent with enzyme activity data. In contrast, double null Bmp1/Tll1 cells produced predominantly unprocessed 50-kDa pro-lysyl oxidase and had lysyl oxidase enzyme activity diminished by 70% compared with wild type, Bmp1-null, and Tll1-null cells. Thus, the combination of BMP-1/mTLD and mTLL-1 is shown to be responsible for the majority of processing leading to activation of lysyl oxidase by murine embryonic fibroblasts, whereas in vitro studies identify pro-lysyl oxidase as the first known substrate for mTLL-2.

High frequency of ras mutations in forestomach and lung tumors of B6C3F1 mice exposed to 1-amino-2,4-dibromoanthraquinone for 2 years.

1-Amino-2,4-dibromoanthraquinone (ADBAQ) is an anthraquinone-derived vat dye, and a potent carcinogen in laboratory animals. In a 2-year study with dietary exposure to 10,000 or 20,000 ppm ADBAQ, increased incidence of forestomach and lung tumors were observed in B6C3F1 mice. The present study indentified genetic alterations in H-ras and K-ras proto-oncogenes in ADBAQ-induced tumors. Point mutations in ras proto-oncogenes were identified by restriction fragment length polymorphism, single-stranded conformational polymorphism analysis and cycle sequencing of polymerase chain reaction-amplified DNA isolated from paraffin-embedded squamous cell papillomas and carcinomas in the forestomach, and alveolar/bronchiolar adenomas and carcinomas in the lung. A higher frequency of ras mutations was identified in ADBAQ-induced forestomach (23/32, 72%) and lung tumors (16/23, 70%) than in spontaneous forestomach (4/11, 36%) and lung tumors (26/86, 30%). H-ras codon 61 CTA mutations were detected in (4/8, 50%) ADBAQ-induced forestomach squamous cell papillomas and (10/24, 42%) squamous cell carcinomas, but not in the spontaneous forestomach tumors examined. H-ras codon 61 CGA mutation (6/24, 25%) was also detected in ADBAQ-induced forestomach squamous cell carcinomas. K-ras codon 61 A to T transversions and A to G transitions were prominent in ADBAQ-induced lung alveolar/bronchiolar adenomas and alveolar/bronchiolar carcinomas. The major finding of A to T transversions or A to G transitions in forestomach and lung tumors suggests that ADBAQ or its metabolites target adenine bases in the ras proto-oncogenes and that these mutations play a dominant role in multi-organ