Ligand-Specific Transcriptional Mechanisms Underlie Aryl Hydrocarbon Receptor-Mediated Developmental Toxicity of Oxygenated PAHs.

Polycyclic aromatic hydrocarbons (PAHs) are priority environmental contaminants that exhibit mutagenic, carcinogenic, proinflammatory, and teratogenic properties. Oxygen-substituted PAHs (OPAHs) are formed during combustion processes and via phototoxidation and biological degradation of parent (unsubstituted) PAHs. Despite their prevalence both in contaminated industrial sites and in urban air, OPAH mechanisms of action in biological systems are relatively understudied. Like parent PAHs, OPAHs exert structure-dependent mutagenic activities and activation of the aryl hydrocarbon receptor (AHR) and cytochrome p450 metabolic pathway. Four-ring OPAHs 1,9-benz-10-anthrone (BEZO) and benz(a)anthracene-7,12-dione (7,12-B[a]AQ) cause morphological aberrations and induce markers of oxidative stress in developing zebrafish with similar potency, but only 7,12-B[a]AQ induces robust Cyp1a protein expression. We investigated the role of the AHR in mediating the toxicity of BEZO and 7,12-B[a]AQ, and found that knockdown of AHR2 rescued developmental effects caused by both compounds. Using RNA-seq and molecular docking, we identified transcriptional responses that precede developmental toxicity induced via differential interaction with AHR2. Redox-homeostasis genes were affected similarly by these OPAHs, while 7,12-B[a]AQ preferentially activated phase 1 metabolism and BEZO uniquely decreased visual system genes. Analysis of biological functions and upstream regulators suggests that BEZO is a weak AHR agonist, but interacts with other transcriptional regulators to cause developmental toxicity in an AHR-dependent manner. Identifying ligand-dependent AHR interactions and signaling pathways is essential for understanding toxicity of this class of environmentally relevant compounds.

Hip joint replacement surgery for idiopathic osteoarthritis aggregates in families.

In order to determine whether there is a genetic component to hip or knee joint failure due to idiopathic osteoarthritis (OA), we invited patients (probands) undergoing hip or knee arthroplasty for management of idiopathic OA to provide detailed family histories regarding the prevalence of idiopathic OA requiring joint replacement in their siblings. We also invited their spouses to provide detailed family histories about their siblings to serve as a control group. In the probands, we confirmed the diagnosis of idiopathic OA using American College of Rheumatology criteria. The cohorts included the siblings of 635 probands undergoing total hip replacement, the siblings of 486 probands undergoing total knee replacement, and the siblings of 787 spouses. We compared the prevalence of arthroplasty for idiopathic OA among the siblings of the probands with that among the siblings of the spouses, and we used logistic regression to identify independent risk factors for hip and knee arthroplasty in the siblings. Familial aggregation for hip arthroplasty, but not for knee arthroplasty, was observed after controlling for age and sex, suggesting a genetic contribution to end-stage hip OA but not to end-stage knee OA. We conclude that attempts to identify genes that predispose to idiopathic OA resulting in joint failure are more likely to be successful in patients with hip OA than in those with knee OA.

Regulation of the strypsin-related proteinase ISP2 by progesterone in endometrial gland epithelium during implantation in mice.

Hormones prepare the uterus for the arrival and subsequent invasion of the embryo during pregnancy. Extracellular matrix-degrading proteinases and their inhibitors are involved in this integration process. Recent genetic evidence indicates that there is redundancy within the implantation proteinase cascade, indicating that additional proteinases may be involved. Recently, we described a novel implantation serine proteinase (ISP1) gene that encodes the embryo-derived enzyme strypsin, which is necessary for blastocyst hatching in vitro and the initiation of invasion. The evidence presented in the present study indicates that a second proteinase secreted from the uterus also participates in lysis of the zona pellucida. A second implantation serine proteinase gene (ISP2) was isolated, which encodes a related secreted tryptase expressed specifically within uterine endometrial glands. In pseudopregnancy, ISP2 gene expression is dependent on progesterone priming and is inhibited by the antiprogestin RU486. On the basis of similarities between ISP2 gene expression and that of a progesterone-regulated luminal proteinase associated with lysis of the zona pellucida, it is possible that the strypsin-related protein, ISP2, may encode a zona lysin proteinase.

Complications of osteotomies in severe cerebral palsy.

Seventy-nine consecutive children with cerebral palsy who underwent osteotomies about the hip for subluxation or dislocation were studied retrospectively to determine risk factors that would correlate with postoperative complications of death, fracture, or decubitus ulcer. Except for the three patients who died, all of the children had > or = 1 year of follow-up. Twenty (25%) patients had at least one complication. Three children died; one at 1 week, one at 2 weeks, and one at 5 months after surgery. Sixteen patients sustained 25 fractures. All were managed with cast or splint immobilization in the clinic. Five patients developed decubitus ulcers requiring > or = 2 weeks of local care, but none required skin grafts or flaps. Complications occurred in 13 (68%) of 19 children with gastrostomies or tracheostomies but in only seven (12%) of the remaining 60 children. Only one (8%) of 13 ambulatory patients had a complication compared with 19 (29%) of 66 nonambulatory patients. In conclusion, ambulatory function correlates well with the risk of complications after osteotomies. A nonambulatory patient with a gastrostomy or tracheostomy is at even greater risk. Fortunately the fractures and ulcers observed in this series healed uneventfully with no operative intervention.

Immobilized pH gradient two-dimensional gel electrophoresis and mass spectrometric identification of cytokine-regulated proteins in ME-180 cervical carcinoma cells.

Two-dimensional (2-D) polyacrylamide gel electrophoresis combined with mass spectrometry is a powerful combination of technologies that allows high resolution separation of proteins and their rapid identification. Immobilized pH gradient (IPG) first-dimensional gels have several advantages over carrier ampholyte isoelectric focusing, including a high degree of reproducibility, good protein spot resolution, and a selection of pH range. Here we demonstrate the utility and efficacy of combining IPG 2-D gel electrophoresis with mass spectrometry to identify interferon-gamma- (IFN) and tumor necrosis factor (TNF)-regulated proteins in ME-180 cervical carcinoma cells. Three cytokine-regulated proteins have been identified, using imidazole-zinc-stained preparative IPG 2-D gels and in-gel tryptic digestion followed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry for determination of peptide masses and sequences: 1) triosephosphate isomerase, a glycolytic pathway enzyme, 2) proteasome subunit C3, which is important in protein degradation, and 3) Ran, a GTP-binding protein important in cell cycle regulation, protein import into the nucleus, and RNA export from the nucleus.

Identification of constitutive and gamma-interferon- and interleukin 4-regulated proteins in the human renal carcinoma cell line ACHN.

The effects of IFN-gamma and interleukin 4 (IL-4) on cell proliferation and two-dimensional gel electrophoretic protein patterns of the human renal carcinoma cell line ACHN were studied. Treatment of the cells with IFN-gamma resulted in a 40-50% decrease in their proliferation. IL-4 treatment resulted in a 30-40% decrease. Treating cells with both cytokines had the same effect as with IFN-gamma alone, thus precluding a synergistic antiproliferative interaction of these two cytokines. To identify IL-4- and IFN-gamma-regulated proteins in ACHN, two-dimensional preparative gel electrophoresis was used, combined with either capillary electrophoresis or high-performance liquid chromatography and either Edman or mass spectrometric sequencing. The following cytokine-induced proteins were identified: tropomyosin, heat shock protein 27, manganese superoxide dismutase, glutathione S-transferase pi, and protein kinase C inhibitor I. Tropomyosin increased 2-fold when cells were treated with IFN-gamma. Levels of heat shock protein 27 increased 2-fold with IL-4, 3-fold with IFN-gamma, and 4-fold when the cytokines were used in combination. Manganese superoxide dismutase increased 3-fold with IFN-gamma but was unaffected by IL-4. Glutathione S-transferase pi increased 3-fold with IFN-gamma. Levels of protein kinase C inhibitor I increased greater than 3-fold with IL-4, 4-fold with IFN-gamma, and 7-fold when both cytokines were used. In addition, the following constitutive ACHN proteins were identified: copper zinc superoxide dismutase, 60S acidic ribosomal protein P2, and a second heat shock protein 27 isoform. These findings help define the biochemical modes of action of IFN-gamma and IL-4 and their potential in the biological therapy of renal cell carcinoma.

Slipped capital femoral epiphysis. Prediction of contralateral involvement.

We performed a retrospective review of the medical records and radiographs of fifty children who had had unilateral slipped capital femoral epiphysis. Our purpose was to determine if there were any epidemiological parameters that were associated with the later development of a contralateral slip. The mean duration of follow-up was thirty-four months (range, twenty-four to sixty-eight months). Physiological maturity, determined with a modified form of the Oxford method for assessment of bone age, strongly correlated with the risk of development of a contralateral slip in patients who were initially seen with a unilateral slip (r = 0.59, p < 0.0005). The scores were determined by four raters; the variation in the predictive value of the scores among the raters was not significant (p = 0.5). The relationship between the score as determined with the modified Oxford method and the risk of contralateral involvement had a linear distribution. The four raters performed a total of 442 observations to determine the modified Oxford scores for the fifty hips. The prevalence of a subsequent slip for the hips that had been assigned a score of 16 points was 85 +/- 15 per cent (95 per cent confidence limit) (seventeen of twenty observations). The risk of contralateral involvement when the score was 21 points was 11 +/- 9 per cent (six of fifty-four observations). A slip did not develop in any hip with a score of 22 points or more (sixty-nine observations). For boys, the age at the time of the initial slip was predictive of a contralateral slip. A contralateral slip developed in all four boys who had been eleven years and seven months old or less at the time of the initial presentation; however, a contralateral slip developed in only nine of the twenty-two boys who had been eleven years and eight months to fourteen years and eleven months old. A contralateral slip did not develop in the three boys who had been fifteen years old or more. There was no association between age and the risk of a contralateral slip in girls.