Serum Buprenorphine Concentrations and Behavioral Activity in Mice After a Single Subcutaneous Injection of Simbadol, Buprenorphine SR-LAB, or Standard Buprenorphine.

Buprenorphine, an analgesic commonly used in rodent surgery, requires repeated dosing every 4 to 6 h in order to provide adequate analgesia. However, redosing requires repeated handling, which may itself cause stress. Buprenorphine SR-LAB, which reportedly maintains serum levels of buprenorphine greater than 1 ng/mL for 48 to 72 h, is commercially available. However, the viscosity of the product and small dosing volumes make accurate dosing a challenge. Simbadol is a concentrated formulation of buprenorphine hydrochloride labeled for use in cats with recommended dosing frequency of every 24 h. We measured serum concentrations over time after a single injection of this product in C57BL/6NCrl mice and compared it to standard buprenorphine (Buprenex) and Buprenorphine SR-LAB. Male and female mice were injected subcutaneously with one of the 3 buprenorphine formulations at a dose of 1 mg/kg at time 0. Groups of mice (n = 8) were euthanized at 1, 4, 8, 12, 16 h for all groups and 24 h for the Simbadol and the Buprenorphine SR-LAB. Liquid chromatography-mass spectrometry (LC-MS/MS) was used to determine concentrations of buprenorphine in each serum sample. High concentrations were observed in both Simbadol and standard buprenorphine groups one hour after injection (>50 ng/mL). These groups had similar buprenorphine concentration curves, including rates of decline. The standard buprenorphine group had mean concentrations less than 1 ng/mL by 12 h and the Simbadol group by 16 h. In contrast, the Buprenorphine SR-LAB group remained above the 1 ng/mL therapeutic threshold throughout the 24 h. In addition, clinical signs, including increased activity, that lasted for up to an hour after the injection in the Simbadol and standard buprenorphine groups. We conclude that Simbadol does not offer dosing advantages over the standard buprenorphine formulation when given at 1 mg/kg. Buprenorphine SR-LAB maintained a steady concentration of buprenorphine above 1 ng/mL for at least 24 h, and as such is a superior choice for providing long-term analgesia.

Evaluation of 2-methoxy-4-nitroaniline (MNA) in hypersensitivity, 14-day subacute, reproductive, and genotoxicity studies.

2-Methoxy-4-nitroaniline (MNA), an intermediate in the synthesis of azo dyes used in textiles and paints, is structurally similar to carcinogenic anilines. Human exposure occurs primarily in the occupational setting through handling of dye dust, and through use and disposal of MNA-containing products. MNA has been reported to induce contact hypersensitivity in a human, myocardial necrosis in rats, and bacterial mutagenicity. This study assessed the subacute toxicity, genotoxicity, contact hypersensitivity, and reproductive toxicity of MNA in rodents in an effort to more fully characterize its toxicological profile. B6C3F1/N mice were exposed to 0, 650, 1250, 2500, 5000, or 10,000 ppm MNA by dosed feed for 14-days to evaluate subacute toxicity and histopathological endpoints. In female mice, decreased body weight (13.5 %) and absolute kidney weight (14.8 %), compared to control, were observed at 10,000 ppm MNA; increased relative liver weight (10-12 %), compared to control, occurred at 5,000-10,000 ppm MNA. In male mice, absolute (15 %) and relative liver weights (9-13 %) were increased at 2,500-5,000 ppm and 1250-10,000 ppm MNA, compared to control, respectively. In both sexes of mice, minimal elevations of hemosiderin pigmentation (a breakdown product of erythrocytes), relative to control, were observed in the liver (10,000 ppm); minimal to moderate elevations of hemosiderin pigmentation (5,000-10,000 ppm) and minimal increases in hematopoietic cell proliferation occurred in the spleen (≥ 1250 ppm). In a reproductive toxicity study, timed-mated female Harlan Sprague Dawley rats were exposed to 0-10,000 ppm MNA by dosed feed from gestation day 6 through postnatal day (PND) 21. Decreases in mean litter weights were observed at 5000 ppm MNA, compared to control, beginning at PND1. To evaluate potential contact hypersensitivity, MNA (2.5-50 %, in dimethylformamide) was applied to the dorsa of both ears of female Balb/c mice once daily for three days. The increase observed in lymph node cell proliferation (10-50 % increase in thymidine uptake compared to control) did not reproducibly achieve the Sensitization Index (SI) 3 level, and there was no ear swelling evident following sensitization with 10-50 % MNA and challenge with 25 % MNA in the mouse ear swelling test. In bacterial mutagenicity assays, MNA (250-1000 µg/plate) induced significant increases, compared to control, in mutant colonies with and without metabolic activation enzymes in Salmonella typhimurium strains TA100 and TA98. These data indicate that MNA is genotoxic, and may induce erythrocyte damage and reactive phagocytosis by macrophages in the liver and spleen.

Evaluation of the genotoxicity of cell phone radiofrequency radiation in male and female rats and mice following subchronic exposure.

The National Toxicology Program tested two common radiofrequency radiation (RFR) modulations emitted by cellular telephones in a 2-year rodent cancer bioassay that included interim assessments of additional animals for genotoxicity endpoints. Male and female Hsd:Sprague Dawley SD rats and B6C3F1/N mice were exposed from Gestation day 5 or Postnatal day 35, respectively, to code division multiple access (CDMA) or global system for mobile modulations over 18 hr/day, at 10-min intervals, in reverberation chambers at specific absorption rates of 1.5, 3, or 6 W/kg (rats, 900 MHz) or 2.5, 5, or 10 W/kg (mice, 1,900 MHz). After 19 (rats) or 14 (mice) weeks of exposure, animals were examined for evidence of RFR-associated genotoxicity using two different measures. Using the alkaline (pH > 13) comet assay, DNA damage was assessed in cells from three brain regions, liver cells, and peripheral blood leukocytes; using the micronucleus assay, chromosomal damage was assessed in immature and mature peripheral blood erythrocytes. Results of the comet assay showed significant increases in DNA damage in the frontal cortex of male mice (both modulations), leukocytes of female mice (CDMA only), and hippocampus of male rats (CDMA only). Increases in DNA damage judged to be equivocal were observed in several other tissues of rats and mice. No significant increases in micronucleated red blood cells were observed in rats or mice. In conclusion, these results suggest that exposure to RFR is associated with an increase in DNA damage. Environ. Mol. Mutagen. 61:276-290, 2020. © 2019 Wiley Periodicals, Inc.

Bisphenol A induces human uterine leiomyoma cell proliferation through membrane-associated ERα36 via nongenomic signaling pathways.

The role of ERα36 in regulating BPA's effects and its potential as a risk factor for human uterine fibroids were evaluated. BPA at low concentrations (10-6 µM - 10 µM) increased proliferation by facilitating progression of hormonally regulated, immortalized human uterine leiomyoma (ht-UtLM; fibroid) cells from G0-G1 into S phase of the cell cycle; whereas, higher concentrations (100 µM-200 µM) decreased growth. BPA upregulated ERα36 gene and protein expression, and induced increased SOS1 and Grb2 protein expression, both of which are mediators of the MAPKp44/42/ERK1/2 pathway. EGFR (pEGFR), Ras, and MAPKp44/42 were phosphorylated with concurrent Src activation in ht-UtLM cells within 10 min of BPA exposure. BPA enhanced colocalization of phosphorylated Src (pSrc) to ERα36 and coimmunoprecipitation of pSrc with pEGFR. Silencing ERα36 with siERα36 abolished the above effects. BPA induced proliferation in ht-UtLM cells through membrane-associated ERα36 with activation of Src, EGFR, Ras, and MAPK nongenomic signaling pathways.

Mutational analysis of pentabrominated diphenyl-induced hepatocellular tumors in rats and mice, tissue levels of PBDE congeners in rats and mice, and AhR genotyping of Wistar Han rats.

This article describes data related to the research article entitled "Carcinogenic activity of pentabrominated diphenyl ether mixture (DE-71) in rats and mice" (Dunnick et al., 2018). PBDE-induced hepatocellular tumors harbored Hras and Ctnnb1 mutations and the methods for these studies are provided. Tissue levels of PBDE congeners in rats and mice after oral exposure to PBDE mixture increased with increasing dose of PBDE. There was no correlation between AhR status and the incidence of hepatocellular tumors in female Wistar Han rats. This manuscript provides additional information on the methods for conducting mutational analysis, PBDE tissue level determinations, and AhR genotyping.

The Life Cycle of the Uterine Fibroid Myocyte.

PURPOSE OF REVIEW: Uterine fibroids are common benign tumors of women in the USA and worldwide, yet the biological nature and pathogenesis of these tumors remain largely unknown. This review presents our view of the stages in the life cycle of a subset of uterine fibroid myocytes, introduces hypothetical concepts and morphological data to explain these changes, and relates these changes in individual myocytes to the phases of fibroid tumor development. RECENT FINDINGS: The observations gained from light and electron microscopic, immunohistochemical, and morphometric studies in our laboratory have led to the hypothesis that fibroid changes over time may relate to the excessive production of collagen by phenotypically transformed myocytes. This accumulation of collagen results in decreased microvessel density, followed by myocyte injury and atrophy, with eventual senescence and involution through ischemic cellular degeneration and inanition. SUMMARY: Uterine leiomyomas, or fibroids, are characterized by two histologic features-proliferation of myocytes and production of an extracellular collagenous matrix. In the larger tumors, the collagenous matrix is often abundant. Within those regions in which the accumulating collagen is excessive, the myocytes are progressively separated from their blood supply, resulting in myocyte atrophy and eventually cell death. It is within these hypocellular, hyalinized areas that the complete lifecycle of the fibroid myocyte is realized. It begins with the phenotypic transformation of a contractile cell to one characterized by proliferation and collagen synthesis, progresses through an intermediate stage of atrophy related to interstitial ischemia, and eventuates in cell death due to inanition. Lastly, resorption of inanotic cells appears to occur by a non-phagocytic, presumably enzymatic process of degradation and recycling that we refer to as reclamation.

Evaluation of Prenatal Exposure to Bisphenol Analogues on Development and Long-Term Health of the Mammary Gland in Female Mice.

BACKGROUND: Continued efforts to phase out bisphenol A (BPA) from consumer products have been met with the challenges of finding safer alternatives. OBJECTIVES: This study aimed to determine whether early-life exposure to BPA and its related analogues, bisphenol AF (BPAF) and bisphenol S (BPS), could affect female pubertal mammary gland development and long-term mammary health in mice. METHODS: Timed pregnant CD-1 mice were exposed to vehicle, BPA (0.5, 5, 50 mg/kg), BPAF (0.05, 0.5, 5 mg/kg), or BPS (0.05, 0.5, 5 mg/kg) via oral gavage between gestation days 10­17. Mammary glands were collected from resulting female offspring at postnatal day (PND) 20, 28, 35, and 56, and at 3, 8, and 14 months for whole mount, histopathological evaluation, and quantitative real-time polymerase chain reaction (qPCR); serum steroid concentrations were also measured at these time points. RESULTS: In the bisphenol-exposed mice, accelerated mammary gland development was evident during early puberty and persisted into adulthood. By late adulthood, mammary glands from bisphenol-exposed female offspring exhibited adverse morphology in comparison with controls; most prominent were undifferentiated duct ends, significantly more lobuloalveolar hyperplasia and perivascular inflammation, and various tumors, including adenocarcinomas. Effects were especially prominent in the BPAF 5 mg/kg and BPS 0.5 mg/kg groups. Serum steroid concentrations and mammary mRNA levels of Esr1, Pgr, Ar, and Gper1 were similar to controls. CONCLUSIONS: These data demonstrate that prenatal exposure of mice to BPAF or BPS induced precocious development of the mammary gland, and that siblings were significantly more susceptible to spontaneous preneoplastic epithelial lesions and inflammation, with an incidence greater than that observed in vehicle- and BPA-exposed animals. https://doi.org/10.1289/EHP3189.

Erratum: "Diversity Outbred Mice Identify Population-Based Exposure Thresholds and Genetic Factors that Influence Benzene-Induced Genotoxicity".

[This corrects the article DOI: http://dx.doi.org/10.1289/ehp.1408202.].

Effect of cell phone radiofrequency radiation on body temperature in rodents: Pilot studies of the National Toxicology Program's reverberation chamber exposure system.

Radiofrequency radiation (RFR) causes heating, which can lead to detrimental biological effects. To characterize the effects of RFR exposure on body temperature in relation to animal size and pregnancy, a series of short-term toxicity studies was conducted in a unique RFR exposure system. Young and old B6C3F1 mice and young, old, and pregnant Harlan Sprague-Dawley rats were exposed to Global System for Mobile Communication (GSM) or Code Division Multiple Access (CDMA) RFR (rats = 900 MHz, mice = 1,900 MHz) at specific absorption rates (SARs) up to 12 W/kg for approximately 9 h a day for 5 days. In general, fewer and less severe increases in body temperature were observed in young than in older rats. SAR-dependent increases in subcutaneous body temperatures were observed at exposures ≥6 W/kg in both modulations. Exposures of ≥10 W/kg GSM or CDMA RFR induced excessive increases in body temperature, leading to mortality. There was also a significant increase in the number of resorptions in pregnant rats at 12 W/kg GSM RFR. In mice, only sporadic increases in body temperature were observed regardless of sex or age when exposed to GSM or CDMA RFR up to 12 W/kg. These results identified SARs at which measurable RFR-mediated thermal effects occur, and were used in the selection of exposures for subsequent toxicology and carcinogenicity studies. Bioelectromagnetics. 39:190-199, 2018. © 2018 The Authors. Bioelectromagnetics Published by Wiley Periodicals, Inc.

Evaluation of the respiratory tract toxicity of ortho-phthalaldehyde, a proposed alternative for the chemical disinfectant glutaraldehyde.

ortho-Phthalaldehyde (OPA) is a high-level chemical disinfectant that is commonly used for chemical sterilization of dental and medical instruments as an alternative to glutaraldehyde, a known skin and respiratory sensitizer. Concern for safe levels of human exposure remains due to a lack of toxicity data as well as human case reports of skin and respiratory sensitization following OPA exposure. The present study evaluated the inhalational toxicity of OPA in Harlan Sprague-Dawley rats and B6C3F1/N mice. Groups of 10 male and female rats and mice were exposed to OPA by whole-body inhalation for 3 months at concentrations of 0 (control), 0.44, 0.88, 1.75, 3.5, or 7.0 ppm. Rats and mice developed a spectrum of lesions at sites of contact throughout the respiratory tract (nose, larynx, trachea, lung), as well as in the skin and eye, consistent with a severe irritant response. In general, histologic lesions (necrosis, inflammation, regeneration, hyperplasia and metaplasia) occurred at deeper sites within the respiratory tract with increasing exposure concentration. As a first site of contact, the nose exhibited the greatest response to OPA exposure and resulted in an increased incidence, severity and variety of lesions compared to a previous study of glutaraldehyde exposure at similar exposure concentrations. This increased response in the nasal cavity, combined with extensive lesions throughout the respiratory tract, provides concern for use of OPA as a replacement for glutaraldehyde as a high-level disinfectant.

Dose-response assessment of the dermal toxicity of Virginia cedarwood oil in F344/N rats and B6C3F1/N mice.

Virginia cedarwood oil is widely used as a fragrance material in household and personal products and as a naturally derived pesticide alternative. Due to conflicting literature on dermal exposures in animals and humans, concern for safe levels of human exposure remains. The present study evaluated the toxicity of cedarwood oil applied dermally to F344/N rats and B6C3F1/N mice for 13 weeks. Groups of 10 male and female rats and mice received no treatment (untreated control) or were administered cedarwood oil in 95% aqueous ethanol dermally at concentrations ranging from 0% (vehicle control), 6.25%, 12.5%, 25%, 50%, and 100% (undiluted). Rats and mice developed extensive skin lesions at the site of application. Benchmark dose modeling (BMD) was performed for the significantly increased skin lesions observed in the rat, to provide perspective for risk assessment applications. Benchmark dose modeling levels (BMDL) of 0.65 to 2.1% and 1.2 to 4.4% (equivalent to 13 to 42 mg/kg and 24 to 48 mg/kg, respectively) cedarwood oil were calculated for the most sensitive endpoint of epidermal hyperplasia in female rats and chronic active inflammation in male rats, respectively. These BMDL levels coincide with reported use levels in cosmetics and pesticides, raising the concern for human exposure.

Evaluation of buprenorphine hydrochloride Pluronic(®) gel formulation in male C57BL/6NCrl mice.

Providing adequate analgesia while minimizing handling and stress post-surgery can be challenging. Recently, there have been commercial products made available for providing long acting analgesia in rodents. However, we find there are limitations for use in mice due to the viscosity of the product and the small dosing volumes needed. This project evaluated an in-house compounded formulation of buprenorphine easily made in the laboratory using pharmaceutical grade products. The release of buprenorphine was evaluated when compounded with two types of hydrogels (Pluronic(®) F-127 and F-68). Mice given buprenorphine in hydrogel (BP) demonstrated higher serum levels of buprenorphine for a longer period of time compared to mice given standard buprenorphine (Bup). However, the rate of decline in serum levels between the groups was similar; thus, it is more likely that the higher buprenorphine concentration seen in the BP group is due to the higher dose of buprenorphine given, rather than a slower release of product. Feed consumption was decreased in both groups one day after dosing; however, there was no difference in body weights. Increased activity in the open field was observed with both buprenorphine formulations, and lipemia was observed in mice given BP which persisted to at least 96 h. Based on our results, we conclude that this formulation did not sustain the release of buprenorphine or eliminate the increased activity commonly seen in mice given buprenorphine. In addition, the lipemia may confound research parameters, especially in cardiac studies and lipid metabolism studies. Therefore, we cannot recommend this formulation for use.

Differences in the Rate of in Situ Mammary Gland Development and Other Developmental Endpoints in Three Strains of Female Rat Commonly Used in Mammary Carcinogenesis Studies: Implications for Timing of Carcinogen Exposure.

The potential of chemicals to alter susceptibility to mammary tumor formation is often assessed using a carcinogen-induced study design in various rat strains. The rate of mammary gland (MG) development must be considered so that the timing of carcinogen administration is impactful. In this study, in situ MG development was assessed in females of the Harlan Sprague-Dawley (Hsd:SD), Charles River Sprague-Dawley (Crl:SD), and Charles River Long-Evans (Crl:LE) rat strains at postnatal days 25, 33, and 45. Development was evaluated by physical assessment of growth parameters, developmental scoring, and quantitative morphometric analysis. Although body weight (BW) was consistently lower and day of vaginal opening (VO) occurred latest in female Hsd:SD rats, they exhibited accelerated pre- and peripubertal MG development compared to other strains. Glands of Crl:SD and Crl:LE rats exhibited significantly more terminal end buds (TEBs) and TEB/mm than Hsd:SD rats around the time of VO. These data suggest a considerable difference in the rate of MG development across commonly used strains, which is independent of BW and timing of VO. In mammary tumor induction studies employing these strains, administration of the carcinogen should be timed appropriately, based on strain, to specifically target the peak of TEB occurrence.

Epigenetic regulation of transcription factor promoter regions by low-dose genistein through mitogen-activated protein kinase and mitogen-and-stress activated kinase 1 nongenomic signaling.

BACKGROUND: The phytoestrogen, genistein at low doses nongenomically activates mitogen-activated protein kinase p44/42 (MAPKp44/42) via estrogen receptor alpha (ERα) leading to proliferation of human uterine leiomyoma cells. In this study, we evaluated if MAPKp44/42 could activate downstream effectors such as mitogen- and stress-activated protein kinase 1 (MSK1), which could then epigenetically modify histone H3 by phosphorylation following a low dose (1 µg/ml) of genistein. RESULTS: Using hormone-responsive immortalized human uterine leiomyoma (ht-UtLM) cells, we found that genistein activated MAPKp44/42 and MSK1, and also increased phosphorylation of histone H3 at serine10 (H3S10ph) in ht-UtLM cells. Colocalization of phosphorylated MSK1 and H3S10ph was evident by confocal microscopy in ht-UtLM cells (r = 0.8533). Phosphorylation of both MSK1and H3S10ph was abrogated by PD98059 (PD), a MEK1 kinase inhibitor, thereby supporting genistein's activation of MSK1 and Histone H3 was downstream of MAPKp44/42. In proliferative (estrogenic) phase human uterine fibroid tissues, phosphorylated MSK1 and H3S10ph showed increased immunoexpression compared to normal myometrial tissues, similar to results observed in in vitro studies following low-dose genistein administration. Real-time RT-PCR arrays showed induction of growth-related transcription factor genes, EGR1, Elk1, ID1, and MYB (cMyb) with confirmation by western blot, downstream of MAPK in response to low-dose genistein in ht-UtLM cells. Additionally, genistein induced associations of promoter regions of the above transcription factors with H3S10ph as evidenced by Chromatin Immunoprecipitation (ChIP) assays, which were inhibited by PD. Therefore, genistein epigenetically modified histone H3 by phosphorylation of serine 10, which was regulated by MSK1 and MAPK activation. CONCLUSION: Histone H3 phosphorylation possibly represents a mechanism whereby increased transcriptional activation occurs following low-dose genistein exposure.

A High Concentration of Genistein Induces Cell Death in Human Uterine Leiomyoma Cells by Autophagy.

Genistein, an estrogenic, soy-derived isoflavone, may play a protective role against hormone-related cancers. We have reported that a high concentration of genistein inhibits cell proliferation and induces apoptosis in human uterine smooth muscle cells, but not in leiomyoma (fibroid) cells. To better understand the differential cell death responses of normal and tumor cells to a high concentration of genistein, we treated uterine smooth muscle cells and uterine leiomyoma cells with 50 µg/ml of genistein for 72 h and 168 h, and assessed for mediators of apoptosis, cytotoxicity and autophagy. We found that leiomyoma cells had increased protection from apoptosis by expressing an increased ratio of Bcl-2: bak at 72 h and 168 h; however, in smooth muscle cells, the Bcl-2: bak ratio was decreased at 72 h, but significantly rebounded by 168 h. The apoptosis extrinsic factors, Fas ligand and Fas receptor, were highly expressed in uterine smooth muscle cells following genistein treatment at both time points as evidenced by confocal microscopy. This was not seen in the uterine leiomyoma cells; however, cytotoxicity as indicated by elevated lactate dehydrogenase levels was significantly enhanced at 168 h. Increased immunoexpression of an autophagy/autophagosome marker was also observed in the leiomyoma cells, although minimally present in smooth muscle cells at 72 h. Ultrastructurally, there was evidence of autophagic vacuoles in the leiomyoma cells; whereas, the normal smooth muscle cells showed nuclear fragmentation indicative of apoptosis. In summary, our data show differential cell death pathways induced by genistein in tumor and normal uterine smooth muscle cells, and suggest novel cell death pathways that can be targeted for preventive and intervention strategies for inhibiting fibroid tumor cell growth in vivo.

Immunotoxic effects of sodium tungstate dihydrate on female B6C3F1/N mice when administered in drinking water.

Tungsten is a naturally occurring, high-tensile strength element that has been used in a number of consumer products. Tungsten has been detected in soil, waterways, groundwater, and human tissue and body fluids. Elevated levels of tungsten in urine were reported for populations exposed to tungstate in drinking water in areas where natural tungsten formations were prevalent. Published reports indicated that sodium tungstate may modulate hematopoiesis, immune cell populations, and immune responses in rodent models. The objective of this study was to assess potential immunotoxicity of sodium tungstate dihydrate (STD), a drinking water contaminant. Female B6C3F1/N mice received 0-2000 mg STD/L in their drinking water for 28 d, and were evaluated for effects on immune cell populations in spleen and bone marrow, and humoral-mediated, cell-mediated, and innate immunity. Three different parameters of cell-mediated immunity were similarly affected at 1000 mg STD/L. T-cell proliferative responses against allogeneic leukocytes and anti-CD3 were decreased 32%, and 21%, respectively. Cytotoxic T-lymphocyte activity was decreased at all effector:target cell ratios examined. At 2000 mg STD/L, the absolute numbers of CD3(+) T-cell progenitor cells in bone marrow were increased 86%, but the alterations in B-lymphocyte and other progenitor cells were not significant. There were no effects on bone marrow DNA synthesis or colony forming capabilities. STD-induced effects on humoral-mediated immunity, innate immunity, and splenocyte sub-populations were limited. Enhanced histopathology did not detect treatment-related lesions in any of the immune tissues. These data suggest exposure to STD in drinking water may adversely affect cell-mediated immunity.

Disruption of estrogen homeostasis as a mechanism for uterine toxicity in Wistar Han rats treated with tetrabromobisphenol A.

Chronic oral treatment of tetrabromobisphenol A (TBBPA) to female Wistar Han rats resulted in increased incidence of cell proliferation at 250mg/kg and tumor formation in the uterus at higher doses. The present study was designed to test the hypothesis that disruption of estrogen homeostasis was a major mode-of-action for the observed effects. Biological changes were assessed in serum, liver, and the proximal (nearest the cervix) and distal (nearest the ovaries) sections of the uterine horn of Wistar Han rats 24h following administration of the last of five daily oral doses of 250mg/kg. Expression of genes associated with receptors, biosynthesis, and metabolism of estrogen was altered in the liver and uterus. TBBPA treatment also resulted in changes in expression of genes associated with cell division and growth. Changes were also observed in the concentration of thyroxine in serum and in expression of genes in the liver and uterus associated with thyroid hormone receptors. Differential expression of some genes was tissue-dependent or specific to tissue location in the uterus. The biological responses observed in the present study support the hypothesis that perturbation of estrogen homeostasis is a major mode-of-action for TBBPA-mediated cell proliferation and tumorigenesis previously observed in the uterus of TBBPA-treated Wistar Han rats.

Effects of in Utero Exposure to Arsenic during the Second Half of Gestation on Reproductive End Points and Metabolic Parameters in Female CD-1 Mice.

BACKGROUND: Mice exposed to high levels of arsenic in utero have increased susceptibility to tumors such as hepatic and pulmonary carcinomas when they reach adulthood. However, the effects of in utero arsenic exposure on general physiological functions such as reproduction and metabolism remain unclear. OBJECTIVES: We evaluated the effects of in utero exposure to inorganic arsenic at the U.S. Environmental Protection Agency (EPA) drinking water standard (10 ppb) and at tumor-inducing levels (42.5 ppm) on reproductive end points and metabolic parameters when the exposed females reached adulthood. METHODS: Pregnant CD-1 mice were exposed to sodium arsenite [none (control), 10 ppb, or 42.5 ppm] in drinking water from gestational day 10 to birth, the window of organ formation. At birth, exposed offspring were fostered to unexposed dams. We examined reproductive end points (age at vaginal opening, reproductive hormone levels, estrous cyclicity, and fertility) and metabolic parameters (body weight changes, hormone levels, body fat content, and glucose tolerance) in the exposed females when they reached adulthood. RESULTS: Arsenic-exposed females (10 ppb and 42.5 ppm) exhibited early onset of vaginal opening. Fertility was not affected when females were exposed to the 10-ppb dose. However, the number of litters per female was decreased in females exposed to 42.5 ppm of arsenic in utero. In both 10-ppb and 42.5-ppm groups, arsenic-exposed females had significantly greater body weight gain, body fat content, and glucose intolerance. CONCLUSION: Our findings revealed unexpected effects of in utero exposure to arsenic: exposure to both a human-relevant low dose and a tumor-inducing level led to early onset of vaginal opening and to obesity in female CD-1 mice.

Immunoexpression of Steroid Hormone Receptors and Proliferation Markers in Uterine Leiomyoma and Normal Myometrial Tissues from the Miniature Pig, Sus scrofa.

Uterine leiomyomas in miniature pet pigs occur similarly to those in women with regard to frequency, age, parity, and cycling. Clinical signs, gross, and histologic features of the porcine tumors closely resemble uterine leiomyomas (fibroids) in women. Although fibroids are hormonally responsive in women, the roles of estrogen and progesterone have not been fully elucidated. In this study, immunohistochemistry was used to assess the expression of the steroid hormone receptors, estrogen receptor alpha (ER-α), estrogen receptor beta (ER-ß) and progesterone receptor (PR), and cell proliferation markers, proliferating cell nuclear antigen (PCNA) and Ki-67 in tumor and matched myometrial tissues sampled from miniature pigs. A "quickscore" method was used to determine receptor expression and labeling indices were calculated for the markers. ER-α/ß and PR were localized to the nuclei of smooth muscle cells in both tissues. PR expression was intense and diffuse throughout all tissues, with correlation between tumors and matched myometria. Conversely, ER-α expression was variable between the myometrial and tumor tissues, as well as between animals. ER-ß expression was low. PCNA and Ki-67 were localized to the nucleus and expression varied among tumors; however, normal tissues were overall negative. These findings support further investigation into the use of the miniature pig as a model of fibroids in women.

Kras, Egfr, and Tp53 Mutations in B6C3F1/N Mouse and F344/NTac Rat Alveolar/Bronchiolar Carcinomas Resulting from Chronic Inhalation Exposure to Cobalt Metal.

Rodent lung tumors are morphologically similar to a subtype of human lung adenocarcinomas. The objective of this study was to evaluate Kirsten rat sarcoma oncogene homolog (Kras), epidermal growth factor receptor (Egfr), and tumor protein 53 (Tp53) mutations, which are relevant to human lung cancer, in cobalt metal dust (CMD)-induced alveolar/bronchiolar tumors of B6C3F1/N mice and F344/NTac rats. Kras mutations were detected in 67% (mice) and 31% (rats) of CMD-induced lung tumors and were predominantly exon 1 codon 12 G to T transversions (80% in mice and 57% in rats). Egfr mutations were detected in 17% (both mice and rats) of CMD-induced lung tumors and were predominantly in exon 20 with 50% G to A transitions (mice and rats). Tp53 mutations were detected in 19% (mice) and 23% (rats) of CMD-induced lung tumors and were predominant in exon 5 (mice, 69% transversions) and exon 6 (rats, all transitions). No mutations were observed for these genes in spontaneous lung tumors or normal lungs from untreated controls. Ames assay indicated that CMD is mutagenic in the absence but not in the presence of S9 mix. Thus, the mutation data (G to T transversions) and Ames assay results suggest that oxidative damage to DNA may be a contributing factor in CMD-induced pulmonary carcinogenesis in rodents.