Xenotransplantation models to study the effects of toxicants on human fetal tissues.

Many diseases that manifest throughout the lifetime are influenced by factors affecting fetal development. Fetal exposure to xenobiotics, in particular, may influence the development of adult diseases. Established animal models provide systems for characterizing both developmental biology and developmental toxicology. However, animal model systems do not allow researchers to assess the mechanistic effects of toxicants on developing human tissue. Human fetal tissue xenotransplantation models have recently been implemented to provide human-relevant mechanistic data on the many tissue-level functions that may be affected by fetal exposure to toxicants. This review describes the development of human fetal tissue xenotransplant models for testis, prostate, lung, liver, and adipose tissue, aimed at studying the effects of xenobiotics on tissue development, including implications for testicular dysgenesis, prostate disease, lung disease, and metabolic syndrome. The mechanistic data obtained from these models can complement data from epidemiology, traditional animal models, and in vitro studies to quantify the risks of toxicant exposures during human development.

Differential response to abiraterone acetate and di-n-butyl phthalate in an androgen-sensitive human fetal testis xenograft bioassay.

In utero exposure to antiandrogenic xenobiotics such as di-n-butyl phthalate (DBP) has been linked to congenital defects of the male reproductive tract, including cryptorchidism and hypospadias, as well as later life effects such as testicular cancer and decreased sperm counts. Experimental evidence indicates that DBP has in utero antiandrogenic effects in the rat. However, it is unclear whether DBP has similar effects on androgen biosynthesis in human fetal testis. To address this issue, we developed a xenograft bioassay with multiple androgen-sensitive physiological endpoints, similar to the rodent Hershberger assay. Adult male athymic nude mice were castrated, and human fetal testis was xenografted into the renal subcapsular space. Hosts were treated with human chorionic gonadotropin for 4 weeks to stimulate testosterone production. During weeks 3 and 4, hosts were exposed to DBP or abiraterone acetate, a CYP17A1 inhibitor. Although abiraterone acetate (14 d, 75 mg/kg/d po) dramatically reduced testosterone and the weights of androgen-sensitive host organs, DBP (14 d, 500 mg/kg/d po) had no effect on androgenic endpoints. DBP did produce a near-significant trend toward increased multinucleated germ cells in the xenografts. Gene expression analysis showed that abiraterone decreased expression of genes related to transcription and cell differentiation while increasing expression of genes involved in epigenetic control of gene expression. DBP induced expression of oxidative stress response genes and altered expression of actin cytoskeleton genes.

Maturation of the developing human fetal prostate in a rodent xenograft model.

BACKGROUND: Prostate cancer is the most commonly diagnosed nonskin cancer in men. The etiology of prostate cancer is unknown, although both animal and epidemiologic data suggest that early life exposures to various toxicants, may impact DNA methylation status during development, playing an important role. METHODS: We have developed a xenograft model to characterize the growth and differentiation of human fetal prostate implants (gestational age 12-24 weeks) that can provide new data on the potential role of early life stressors on prostate cancer. The expression of key immunohistochemical markers responsible for prostate maturation was evaluated, including p63, cytokeratin 18, α-smooth muscle actin, vimentin, caldesmon, Ki-67, prostate-specific antigen, estrogen receptor-α, and androgen receptor. Xenografts were separated into epithelial and stromal compartments using laser capture microdissection (LCM), and the DNA methylation status was assessed in >480,000 CpG sites throughout the genome. RESULTS: Xenografts demonstrated growth and maturation throughout the 200 days of post-implantation evaluation. DNA methylation profiles of laser capture microdissected tissue demonstrated tissue-specific markers clustered by their location in either the epithelium or stroma of human prostate tissue. Differential methylated promoter region CpG-associated gene analysis revealed significantly more stromal than epithelial DNA methylation in the 30- and 90-day xenografts. Functional classification analysis identified CpG-related gene clusters in methylated epithelial and stromal human xenografts. CONCLUSION: This study of human fetal prostate tissue establishes a xenograft model that demonstrates dynamic growth and maturation, allowing for future mechanistic studies of the developmental origins of later life proliferative prostate disease.

Clinical evaluation of the FreeStyle Precision Pro system.

BACKGROUND: A new version of international standard (ISO 15197) and CLSI Guideline (POCT12) with more stringent accuracy criteria are near publication. We evaluated the glucose test performance of the FreeStyle Precision Pro system, a new blood glucose monitoring system (BGMS) designed to enhance accuracy for point-of-care testing (POCT). METHODS: Precision, interference and system accuracy with 503 blood samples from capillary, venous and arterial sources were evaluated in a multicenter study. Study results were analyzed and presented in accordance with the specifications and recommendations of the final draft ISO 15197 and the new POCT12. RESULTS: The FreeStyle Precision Pro system demonstrated acceptable precision (CV <5%), no interference across a hematocrit range of 15-65%, and, except for xylose, no interference from 24 of 25 potentially interfering substances. It also met all accuracy criteria specified in the final draft ISO 15197 and POCT12, with 97.3-98.9% of the individual results of various blood sample types agreeing within ±12 mg/dl of the laboratory analyzer values at glucose concentrations <100mg/dl and within ±12.5% of the laboratory analyzer values at glucose concentrations ≥100 mg/dl. CONCLUSIONS: The FreeStyle Precision Pro system met the tighter accuracy requirements, providing a means for enhancing accuracy for point-of-care blood glucose monitoring.

"Man points": masculine capital and young men's health.

OBJECTIVE: Health behaviors are important resources for the development and display of masculine identity. The aim of this mixed-method study was to examine how "masculine capital" is accrued via traditionally masculine behaviors and used to permit nonmasculine behavior. METHODS: An online survey assessing personal importance of gender identity, gender role stereotypes, and beliefs about the gender of various health behaviors was completed by 731 university students. Interviews were conducted with a purposive sample of 16 of these men and women. RESULTS: Quantitative data showed significant positive associations between perceived masculinity and engagement in a greater number of traditionally masculine health behaviors. Such patterns were clearest among young men and women who endorsed gender role stereotypes and gave greater importance to their own gender identity. Qualitative data supported the quantitative data: participants with more traditional gender role beliefs had more strict beliefs about the masculinity of various health behaviors. When asked about their own experiences, many men described having engaged in traditionally masculine health-related behaviors so as to accrue masculine capital or use it to permit nonmasculine (or feminine) behavior. CONCLUSIONS: The novel use of a gender-relations approach in this mixed-method study of young men and women expanded on earlier smaller scale studies of men and masculine capital. The findings add to understanding of the concept of "masculine capital" and suggest how it may aid efforts to better understand and improve young men's health. Young men's concerns about masculinity could be harnessed to encourage healthy "masculine" behavior. However, such approaches may not be effective for men who eschew traditional definitions of masculinity. Furthermore, failure to question socially constructed definitions of gender may reinforce stereotypes that restrict men's and women's opportunities.

Male reprotoxicity and endocrine disruption.

Mammalian reproductive tract development is a tightly regulated process that can be disrupted following exposure to drugs, toxicants, endocrine-disrupting chemicals (EDCs), or other compounds via alterations to gene and protein expression or epigenetic regulation. Indeed, the impacts of developmental exposure to certain toxicants may not be fully realized until puberty or adulthood when the reproductive tract becomes sexually mature and altered functionality is manifested. Exposures that occur later in life, once development is complete, can also disrupt the intricate hormonal and paracrine interactions responsible for adult functions, such as spermatogenesis. In this chapter, the biology and toxicology of the male reproductive tract is explored, proceeding through the various life stages including in utero development, puberty, adulthood, and senescence. Special attention is given to the discussion of EDCs, chemical mixtures, low-dose effects, transgenerational effects, and potential exposure-related causes of male reproductive tract cancers.

Rodent thyroid, liver, and fetal testis toxicity of the monoester metabolite of bis-(2-ethylhexyl) tetrabromophthalate (tbph), a novel brominated flame retardant present in indoor dust.

BACKGROUND: Bis-(2-ethylhexyl) tetrabromophthalate (TBPH) is widely used as a replacement for polybrominated diphenyl ethers (PBDEs) in commercial flame retardant mixtures such as Firemaster 550. It is also used in a commercial mixture called DP 45. Mono-(2-ethyhexyl) tetrabromophthalate (TBMEHP) is a potentially toxic metabolite. OBJECTIVES: We used in vitro and rodent in vivo models to evaluate human exposure and the potential metabolism and toxicity of TBPH. METHODS: Dust collected from homes, offices, and cars was measured for TBPH by gas chromatography followed by mass spectrometry. Pregnant rats were gavaged with TBMEHP (200 or 500 mg/kg) or corn oil on gestational days 18 and 19, and dams and fetuses were evaluated histologically for toxicity. We also assessed TBMEHP for deiodinase inhibition using rat liver microsomes and for peroxisome proliferator-activated receptor (PPAR) α and γ activation using murine FAO cells and NIH 3T3 L1 cells. RESULTS: TBPH concentrations in dust from office buildings (median, 410 ng/g) were higher than in main living areas in homes (median, 150 ng/g). TBPH was metabolized by purified porcine esterases to TBMEHP. Two days of TBMEHP exposure in the rat produced maternal hypothyroidism with markedly decreased serum T3 (3,3´,5-triiodo-l-thyronine), maternal hepatotoxicity, and increased multinucleated germ cells (MNGs) in fetal testes without antiandrogenic effects. In vitro, TBMEHP inhibited deiodinase activity, induced adipocyte differentiation in NIH 3T3 L1 cells, and activated PPARα- and PPARγ-mediated gene transcription in NIH 3T3 L1 cells and FAO cells, respectively. CONCLUSIONS: TBPH a) is present in dust from indoor environments (implying human exposure) and b) can be metabolized by porcine esterases to TBMEHP, which c) elicited maternal thyrotoxic and hepatotoxic effects and d) induced MNGs in the fetal testes in a rat model. In mouse NIH 3T3 L1 preadipocyte cells, TBMEHP inhibited rat hepatic microsome deiodinase activity and was an agonist for PPARs in murine FAO and NIH 3T3 L1 cells.

Human fetal testis xenografts are resistant to phthalate-induced endocrine disruption.

BACKGROUND: In utero exposure to endocrine-disrupting chemicals may contribute to testicular dysgenesis syndrome (TDS), a proposed constellation of increasingly common male reproductive tract abnormalities (including hypospadias, cryptorchidism, hypospermatogenesis, and testicular cancer). Male rats exposed in utero to certain phthalate plasticizers exhibit multinucleated germ cell (MNG) induction and suppressed steroidogenic gene expression and testosterone production in the fetal testis, causing TDS-consistent effects of hypospadias and cryptorchidism. Mice exposed to phthalates in utero exhibit MNG induction only. This disparity in response demonstrates a species-specific sensitivity to phthalate-induced suppression of fetal Leydig cell steroidogenesis. Importantly, ex vivo phthalate exposure of the fetal testis does not recapitulate the species-specific endocrine disruption, demonstrating the need for a new bioassay to assess the human response to phthalates. OBJECTIVES: In this study, we aimed to develop and validate a rat and mouse testis xenograft bioassay of phthalate exposure and examine the human fetal testis response. METHODS: Fetal rat, mouse, and human testes were xenografted into immunodeficient rodent hosts, and hosts were gavaged with a range of phthalate doses over multiple days. Xenografts were harvested and assessed for histopathology and steroidogenic end points. RESULTS: Consistent with the in utero response, phthalate exposure induced MNG formation in rat and mouse xenografts, but only rats exhibited suppressed steroidogenesis. Across a range of doses, human fetal testis xenografts exhibited MNG induction but were resistant to suppression of steroidogenic gene expression. CONCLUSIONS: Phthalate exposure of grafted human fetal testis altered fetal germ cells but did not reduce expression of genes that regulate fetal testosterone biosynthesis.

'That's OK. He's a guy': a mixed-methods study of gender double-standards for alcohol use.

Although drinking and drunkenness have traditionally been considered masculine behaviours, young women's alcohol consumption has increased in recent years. This mixed methods study was conducted to examine the extent to which young people endorse gender double-standards for alcohol use--i.e., less acceptance of drinking and drunkenness in women than men--and how these influence men's and women's alcohol consumption. A sample of 731 English university students completed an online survey of gender role attitudes, beliefs about the gendered nature of alcohol use and recent alcohol consumption. Sixteen participants were then purposively selected for individual interviews: eight women and men with the most egalitarian gender role beliefs, and eight women and men with the least egalitarian beliefs. The two sets of data revealed that although there were few sex differences in actual levels of drinking or drunkenness, gender double-standards for alcohol use persist: beer drinking, binge drinking and public drunkenness tended to be perceived as masculine, and even the most egalitarian respondents were more judgemental of women's drinking. Participants modified their drinking style so as to maintain a desired gender identity. Although gender double-standards could be a focus of interventions to encourage moderate drinking, such approaches could reinforce gender inequalities.

'That's not masculine': masculine capital and health-related behaviour.

In recent years increasing attention has been given to how different masculinities are expressed in young men's health behaviour. To examine whether men can use competence in key health-related masculine domains to compensate for other non-masculine behaviour, group discussions were conducted with men aged 18-21 living in London, England. The analysis revealed the ways in which competence in traditionally masculine health-related domains produces masculine 'capital', which can be used to compensate for non-masculine behaviour in other domains. However, the capacity to trade this capital is limited because different masculine and non-masculine behaviours have different values.

Correlates of parents' reports of acceptability of human papilloma virus vaccination for their school-aged children.

BACKGROUND: Routine human papilloma virus (HPV) vaccination for 12-13-year-old girls will be introduced in the UK from September 2008. The aim of the present study was to identify correlates of parents' anticipated uptake of HPV vaccination for their sons and daughters. METHODS: Self-administered questionnaires were completed by 353 parents of school-aged children living in Brighton and Hove (England). The main outcome measure was anticipated acceptance of HPV vaccination for children. Putative predictors of acceptance of HPV vaccination included general attitudes toward vaccination, beliefs about the impact on adolescent sexual behaviour of vaccines against sexually transmissible infections, and knowledge of HPV and cervical cancer. RESULTS: Multivariate regression revealed that greater perceived benefits of HPV vaccination, greater general belief in the protection offered by vaccination, and greater support for adolescent sexual health services explained substantial proportions of the variance in HPV vaccine acceptability for both sons and daughters. For both sons and daughters, the most important correlate of vaccine acceptability was general belief in the protection offered by vaccination: this variable explained 40-50% of variance. Acceptability of vaccination appeared to improve following the provision of brief information about the links between HPV and cervical cancer and the proposed introduction of HPV vaccination. CONCLUSIONS: Uptake of HPV vaccination may be maximised by: improving attitudes toward the safety and efficacy of childhood vaccinations; countering concerns that provision of sexual health services for young people will encourage promiscuous or unsafe sexual behaviour; and improving knowledge about the role of HPV in cervical cancer aetiology.

Variation of serum creatinine, cystatin C, and creatinine clearance tests in persons with normal renal function.

BACKGROUND: To determine the potential sensitivity of several renal function tests for detecting early changes in renal function, we compared the within-individual (W-I) variation over 5 months of serum creatinine, serum cystatin C, and creatinine clearance. METHODS: On 31 healthy subjects, blood and timed urine specimens were collected once each month to get 6 collections. Creatinine (enzymatic) in serum and urine and cystatin C (immunonephelometric) in serum were measured and glomerular filtration rate (GFR) by creatinine clearance and the Modification of Diet in Renal Disease (MDRD) equation were calculated. To compare W-I variations between different creatinine methods, we also measured creatinine by both enzymatic and kinetic alkaline picrate methods on 15 sets of frozen samples. RESULTS: For the 31 volunteers, the mean W-I variations for serum creatinine (5.8%) and cystatin C (5.4%) were both much lower than the W-I variation of creatinine clearance (18.7%). As expected, the MDRD GFR had a similar W-I variation (6.7%) to that of serum creatinine and its values were markedly different than GFR by creatinine clearance. On the 15 sets of frozen samples, the W-I variation of creatinine measured by the enzymatic method (CV 5.2%) was slightly less than by the picrate method (CV 6.2%). CONCLUSIONS: The low W-I variation of both serum cystatin C and serum creatinine suggests that serial measurements of either would detect a changes in renal function earlier than would GFR by creatinine clearance or MDRD equation, which allows reporting only for GFRs<60 ml/min/1.7 m(2). While we measured only creatinine clearance, the large variability, difficulty, and cost of all clearance measurements make them impractical for routine monitoring of patients.

Validation of a quality assessment system for blood gas and electrolyte testing.

BACKGROUND: A recently-introduced quality assessment system (Intelligent Quality Management: iQM), was evaluated in routine clinical use at four different hospitals. The iQM technology is designed to replace conventional external liquid controls with software, Process Control (PC) Solutions and Calibration Validation components that continually assess the function of the GEM Premier 3000 (GEM) analyzer and automatically initiate and document corrective actions. METHODS: We validated the performance claims of iQM by monitoring quality control (QC) materials at 4 clinical sites while analyzing approximately 10,550 patient samples. We compared iQM-measured QC values to traditional QC results, evaluating the number and type of error flags for patient samples, and used data from control results to calculate the average time to detect an error (ADT) for each analyte. RESULTS: The calculated ADT was approximately 3 min for all analytes except for sodium (17 min), glucose (11 min), and lactate (5.9 min). Precision of control materials in iQM cartridges was better than from external controls run on traditional analyzers. The iQM system detected errors in 0.46% of actual clinical samples. CONCLUSIONS: The findings from our study confirm that (a) iQM precision in a clinical setting is comparable to that found in previous studies done in a research setting, (b) the improved precision of control material on the iQM is likely because the internal control fluids are sealed and not susceptible to exposure from handling, and (c) the system detects and often corrects errors in specific samples that might not be reported by traditional analytical systems.

Development and characterization of on-chip biopolymer membranes.

In lab-on-a-chip applications, filtration is currently performed prior to sample loading or through pre-cast membranes adhered to the substrate. These membranes cannot be patterned to micrometer resolution, and their adhesion may be incompatible with the fabrication process or may introduce contaminants. We have developed an on-chip separation process using a biocompatible polymer that can be patterned and has controllable molecular rejection properties. We spun cast cellulose acetate (CA) membranes directly onto silicon wafers. Characterization of the molecular flux across the membrane showed that molecular weight and charge are major factors contributing to the membranes' rejection characteristics. Altering casting conditions such as polymer concentration in the casting solution and the quenching-bath composition and/or temperature allowed control of the molecular weight cut-off (MWCO). Three MWCOs; 300, 350, and 700 Da have been achieved for non-linear molecules. Molecular shape is also very important as much higher molecular weight single-stranded DNA was electrophoresed across the membranes while heme with a similar negative charge density was rejected. This was due to DNA's small molecular cross section. This is an important result because heme inhibits polymerase chain reactions (PCR) reducing the detection and characterization of DNA from blood samples.

pH effects on measurements of ionized calcium and ionized magnesium in blood.

CONTEXT: It is well known that the concentration of ionized calcium in blood is affected by the pH of the specimen, since hydrogen ions compete with calcium for binding sites on albumin and other proteins. However, the relationship between pH and ionized magnesium concentration is not as well characterized. OBJECTIVE: To determine the effects of pH on ionized magnesium concentration over a wide range of pH values in serum or plasma. DESIGN: Both ionized calcium and ionized magnesium concentrations were measured in 3 sets of samples. (1) Pools of serum or whole blood at different pH values (7.20-7.60) achieved by adding a constant volume of acid or base (diluted solutions of either hydrochloric acid or sodium hydroxide) plus saline. These pools consisted of 2 serum and 3 heparinized whole blood pools collected from leftover blood remaining in clinical specimens in the Clinical Chemistry and Blood Gas Laboratories, respectively, at Duke University Medical Center. (2) Five whole blood specimens obtained from apparently healthy individual donors. (3) Twenty-six whole blood specimens obtained from individual patients (leftover blood from the Blood Gas Laboratory) in which pH was varied by in vitro loss or gain of carbon dioxide. RESULTS: Both ionized calcium and ionized magnesium concentrations decreased as the pH in the specimen increased, indicating the stronger binding of these ions with proteins in the more alkaline environment. CONCLUSION: We conclude that the rate of change of ionized magnesium concentration with pH change (0.12 mmol/L per pH unit) is significantly less than that of ionized calcium (0.36 mmol/L per pH unit). Furthermore, our findings indicate that if adjustment to pH 7.40 is necessary, the ionized magnesium test results need to be adjusted when pH is markedly abnormal, as is sometimes done for ionized calcium.

Activated clotting time systems vary in precision and bias and are not interchangeable when following heparin management protocols during cardiopulmonary bypass.

OBJECTIVE: Our aim was to test the hypothesis that new activated clotting time (ACT) technology, with modifications to instruments and reagents designed to detect earlier clot formation, would be associated with more precise but lower results. A secondary objective was to evaluate the potential impact of any change in ACT measurement on heparin requirements during cardiopulmonary bypass (CPB). METHODS: We compared the precision of two newer ACT systems: Actalyke, Helena Laboratories, Beaumont, TX and Hemochron Response, International Technidyne Corporation, Edison, NJ and assessed their bias with reference to a standard ACT system (Hemochron 801, International Technidyne Corporation, Edison, NJ). Bland-Altman analysis was applied to 81 duplicate samples from 22 patients undergoing CPB or percutaneous coronary interventions (PCI), covering the full clinical range of ACT values. We also estimated the change in heparin dose required to use the Actalyke rather than the Hemochron 801 results, to achieve our target ACT for CPB (480 seconds), and used a mixed model to test for significance. RESULTS: The precision of the Actalyke was superior to the Hemochron Response (mean difference of duplicates +/- 0.1% versus +/- 4.2%). There was no significant bias (p = 0.93) between the results from the standard analyzers (Hemochron 801 and Response), but the results from the modified system (Actalyke) were on average 18% lower than the Hemochron 801 (p < 0.0001). Estimated heparin requirements established that fifty percent of CPB patients would have required additional heparin (5000 to 17500 units), an average increase of 1060 units per patient (p = 0.05), if the Actalyke values were used to guide anticoagulation during CPB. CONCLUSIONS: Our results support the hypothesis that the modified technology (Actalyke) is associated with more precise but lower ACT results. We estimated these lower values would lead to increased heparin dosing during CPB. The impact of this increase on bleeding after cardiac surgery with CPB is controversial and requires further study.