Investigating dimensionality and measurement bias of DSM-5 alcohol use disorder in a representative sample of the largest metropolitan area in South America.

BACKGROUND: Given the recent launch of a new diagnostic classification (DSM-5) for alcohol use disorders (AUD), we aimed to investigate its dimensionality and possible measurement bias in a non-U.S. METHODS: The current analyses were restricted to 948 subjects who endorsed drinking at least one drink per week in the past year from a sample of 5037 individuals. Data came from São Paulo Megacity Project (which is part of World Mental Health Surveys) collected between 2005 and 2007. First, exploratory factor analysis (EFA) was carried out to test for the best dimensional structure for DSM-5-AUD criteria. Then, item response theory (IRT) was used to investigate the severity and discrimination properties of each criterion of DSM-5-AUD. Finally, differential criterion functioning (DCF) were investigated by socio-demographics (income, gender, age, employment status, marital status and education). All analyses were performed in Mplus software taking into account complex survey design features. RESULTS: The best EFA model was a one-dimensional model. IRT results showed that the criteria "Time Spent" and "Given Up" have the highest discrimination and severity properties, while the criterion "Larger/Longer" had the lowest value of severity, but an average value of discrimination. Only female gender had DCF both at criterion- and factor-level, rendering measurement bias. CONCLUSION: This study reinforces the existence of a DSM-5-AUD continuum in the largest metropolitan area of South America, including subgroups that had previously higher rates of alcohol use (lower educational/income levels). Lower DSM-5-AUD scores were found in women.

DSM-5 latent classes of alcohol users in a population-based sample: results from the São Paulo Megacity Mental Health Survey, Brazil.

BACKGROUND: We aimed to identify different categorical phenotypes based upon the DSM-V criteria of alcohol use disorders (AUD) among alcohol users who had at least one drink per week in the past year (n=948). METHODS: Data are from the São Paulo Megacity Mental Health Survey collected in 2005-2007, as part of the World Mental Health Survey Initiative. A latent class analysis of the 11 DSM-5-AUD criteria was performed using Mplus, taking into account complex survey design features. Weighted logistic regression models were used to examine demographic correlates of the DSM-5-AUD latent classes. RESULTS: The best latent-class model was a three-class model. We found a "non-symptomatic class" (69.7%), a "use in larger amounts class" (23.2%), defined by high probability (>70%) of the "use in larger amounts" criterion only, and a "high-moderate symptomatic class" (7.1%), defined by high-moderate probability of all the 11 AUD criteria. Compared to those in the non-symptomatic class, individuals in the "high-moderate symptomatic class" were more likely to have been married, have lower educational attainment and to be unemployed or in non-regular/informal employment. Those on the "use in larger amounts class" were more likely to have been married or never married. CONCLUSION: The two symptomatic classes clearly represented the dimensionality of the new proposed AUD criteria, and could be more specifically targeted by different prevention or treatment strategies. DSM-5-AUD has the advantage of shedding light on risky drinkers included in the "use in larger amounts class", allowing for preventive interventions, which will reach a large number of individuals.

Days out-of-role due to common physical and mental health problems: results from the São Paulo Megacity Mental Health Survey, Brazil.

OBJECTIVES: To investigate the relative importance of common physical and mental disorders with regard to the number of days out-of-role (DOR; number of days for which a person is completely unable to work or carry out normal activities because of health problems) in a population-based sample of adults in the São Paulo Metropolitan Area, Brazil. METHODS: The São Paulo Megacity Mental Health Survey was administered during face-to-face interviews with 2,942 adult household residents. The presence of 8 chronic physical disorders and 3 classes of mental disorders (mood, anxiety, and substance use disorders) was assessed for the previous year along with the number of days in the previous month for which each respondent was completely unable to work or carry out normal daily activities due to health problems. Using multiple regression analysis, we examined the associations of the disorders and their comorbidities with the number of days out-of-role while controlling for socio-demographic variables. Both individual-level and population-level associations were assessed. RESULTS: A total of 13.1% of the respondents reported 1 or more days out-of-role in the previous month, with an annual median of 41.4 days out-of-role. The disorders considered in this study accounted for 71.7% of all DOR; the disorders that caused the greatest number of DOR at the individual-level were digestive (22.6), mood (19.9), substance use (15.0), chronic pain (16.5), and anxiety (14.0) disorders. The disorders associated with the highest population-attributable DOR were chronic pain (35.2%), mood (16.5%), and anxiety (15.0%) disorders. CONCLUSIONS: Because pain, anxiety, and mood disorders have high effects at both the individual and societal levels, targeted interventions to reduce the impairments associated with these disorders have the highest potential to reduce the societal burdens of chronic illness in the São Paulo Metropolitan Area.

Days out-of-role due to common physical and mental health problems: Results from the Sao Paulo Megacity Mental Health Survey, Brazil

OBJECTIVES: To investigate the relative importance of common physical and mental disorders with regard to the number of days out-of-role (DOR; number of days for which a person is completely unable to work or carry out normal activities because of health problems) in a population-based sample of adults in the São Paulo Metropolitan Area, Brazil. METHODS: The São Paulo Megacity Mental Health Survey was administered during face-to-face interviews with 2,942 adult household residents. The presence of 8 chronic physical disorders and 3 classes of mental disorders (mood, anxiety, and substance use disorders) was assessed for the previous year along with the number of days in the previous month for which each respondent was completely unable to work or carry out normal daily activities due to health problems. Using multiple regression analysis, we examined the associations of the disorders and their comorbidities with the number of days out-of-role while controlling for socio-demographic variables. Both individual-level and population-level associations were assessed. RESULTS: A total of 13.1% of the respondents reported 1 or more days out-of-role in the previous month, with an annual median of 41.4 days out-of-role. The disorders considered in this study accounted for 71.7% of all DOR; the disorders that caused the greatest number of DOR at the individual-level were digestive (22.6), mood (19.9), substance use (15.0), chronic pain (16.5), and anxiety (14.0) disorders. The disorders associated with the highest population-attributable DOR were chronic pain (35.2%), mood (16.5%), and anxiety (15.0%) disorders. CONCLUSIONS: Because pain, anxiety, and mood disorders have high effects at both the individual and societal levels, targeted interventions to reduce the impairments associated with these disorders have the highest potential to reduce the societal burdens of chronic illness in the São Paulo Metropolitan Area. .

Estrogen receptors mediate rapid activation of phospholipase C pathway in the rat endometrium.

The aim of the present study was to investigate the activation of rapid signaling events by 17ß-estradiol in the rat uterus. 17ß-Estradiol induced a rapid increase of total [3H]-inositol phosphate accumulation in the whole uterus and endometrium, but not in the myometrium. The effect of 17ß-estradiol in the endometrium was blocked by phospholipase C (PLC) inhibitor (U73122), estrogen receptors antagonist (ICI 182,780), exportin CRM1 inhibitor (leptomycin B) and selective inhibitor of the SRC family of protein tyrosine kinases (PP2). Furthermore, a selective agonist of ESR1 (PPT) and a selective agonist of GPER (G-1) also induced a rapid increase of total [(3)H]-inositol phosphate accumulation in the endometrium. The G-1 effects were blocked by GPER antagonist (G-15). 17ß-Estradiol and G-1 promoted an additive effect on total [3H]-inositol phosphate accumulation. In conclusion, the present results indicate that a rapid activation of the PLC-mediated phosphoinositide hydrolysis occurred in the rat endometrium after 17ß-estradiol stimulation, and this effect was mediated by ESR1 that underwent nuclear export after hormone stimulation, and that GPER activation may play an additive role for this response. These rapid actions might be one of the key steps that mediate the estrogen-dependent activation of cellular events in the endometrium.

Estrogen receptors mediate rapid activation of phospholipase C pathwayin the rat endometrium

The aim of the present study was to investigate the activation of rapid signaling events by 17b-estradiolin the rat uterus. 17b-Estradiol induced a rapid increase of total [3H]-inositol phosphate accumulation inthe whole uterus and endometrium, but not in the myometrium. The effect of 17b-estradiol in the endometriumwas blocked by phospholipase C (PLC) inhibitor (U73122), estrogen receptors antagonist (ICI 182,780), exportin CRM1 inhibitor (leptomycin B) and selective inhibitor of the SRC family of protein tyrosine kinases (PP2). Furthermore, a selective agonist of ESR1 (PPT) and a selective agonist of GPER (G-1) also induced a rapid increase of total [3H]-inositol phosphate accumulation in the endometrium.The G-1 effects were blocked by GPER antagonist (G-15). 17b-Estradiol and G-1 promoted an additive effect on total [3H]-inositol phosphate accumulation. In conclusion, the present results indicate that a rapid activation of the PLC-mediated phosphoinositide hydrolysis occurred in the rat endometrium after 17b-estradiol stimulation, and this effect was mediated by ESR1 that underwent nuclear export after hormonestimulation, and that GPER activation may play an additive role for this response. These rapid actions might be one of the key steps that mediate the estrogen-dependent activation of cellular events in the endometrium.

Environmental toxicants and male reproductive function.

Environmental toxicants, such as cadmium and bisphenol A (BPA) are endocrine disruptors. In utero, perinatal or neonatal exposure of BPA to rats affect the male reproductive function, such as the blood-testis barrier (BTB) integrity. This effect of BPA on BTB integrity in immature rats is likely mediated via a loss of gap junction function at the BTB, failing to coordinate tight junction and anchoring junction function at the site to maintain the immunological barrier integrity. This in turn activates the extracellular signal-regulated kinases 1/2 (Erk1/2) downstream and an increase in protein endocytosis, destabilizing the BTB. The cadmium-induced disruption of testicular dysfunction is mediated initially via its effects on the occludin/ZO-1/focal adhesion kinase (FAK) complex at the BTB, causing redistribution of proteins at the Sertoli-Sertoli cell interface, leading to the BTB disruption. The damaging effects of these toxicants to testicular function are mediated by mitogen-activated protein kinases (MAPK) downstream, which in turn perturbs the actin bundling and accelerates the actin-branching activity, causing disruption of the Sertoli cell tight junction (TJ)-barrier function at the BTB and perturbing spermatid adhesion at the apical ectoplasmic specialization (apical ES, a testis-specific anchoring junction type) that leads to premature release of germ cells from the testis. However, the use of specific inhibitors against MAPK was shown to block or delay the cadmium-induced testicular injury, such as BTB disruption and germ cell loss. These findings suggest that there may be a common downstream p38 and/or Erk1/2 MAPK-based signaling pathway involving polarity proteins and actin regulators that is shared between different toxicants that induce male reproductive dysfunction. As such, the use of inhibitors and/or antagonists against specific MAPKs can possibly be used to "manage" the illnesses caused by these toxicants and/or "protect" industrial workers being exposed to high levels of these toxicants in their work environment.

Expression and signaling of G protein-coupled estrogen receptor 1 (GPER) in rat sertoli cells.

The aim of the present study was to investigate the expression and signaling of the G protein-coupled estrogen receptor 1 (GPER) in cultured immature rat Sertoli cells--in which we have previously described the classical estrogen receptors (ESR1 and ESR2). Expression of GPER in cultured Sertoli cells from 15-day-old rats was detected by RT-PCR and immunoassays. Gper transcripts also were present in testes from 5-, 15-, and 120-day-old rats. Short-term treatment of Sertoli cells with 17beta-estradiol (E2), the GPER agonist G-1, or the ESR antagonist ICI 182,780 (ICI) rapidly activated MAPK3/1 (ERK1/2), even after down-regulation of ESR1 and ESR2, suggesting a role for GPER in the rapid E2 action in these cells. MAPK3/1 phosphorylation induced by ICI or G-1 was blocked by pertussis toxin, selective inhibitor of the SRC family of protein tyrosine kinases, metalloprotease inhibitor, MAP2K1/2 inhibitor, and epidermal growth factor receptor (EGFR) kinase inhibitor. Furthermore, E2, but not G-1, induced up-regulation of cyclin D1 in the Sertoli cells. This effect was blocked by ICI. E2 and G-1 decreased BAX and increased BCL2 expression and these effects were blocked by MAP2K1/2 inhibitor and EGFR kinase inhibitor. The pretreatment with ICI did not block the effect of E2. Taken together, these results indicate that in Sertoli cells 1) GPER-mediated MAPK3/1 activation occurs via EGFR transactivation through G protein beta gamma subunits that promote SRC-mediated metalloprotease-dependent release of EGFR ligands, which bind to EGFR and lead to MAPK3/1 phosphorylation; 2) E2-ESRs play a role in Sertoli cell proliferation; and 3) E2-GPER may regulate gene expression involved with apoptosis. ESR and GPER may mediate actions important for Sertoli cell function and maintenance of normal testis development and homeostasis.

Muscarinic acetylcholine receptor subtypes in the male reproductive tract: expression and function in rat efferent ductules and epididymis.

In mammals, at least five different muscarinic acetylcholine receptor subtypes (mAChRs; M(1)-M(5)) are known to be widely expressed and distributed in different tissues from different species. They mediate distinct physiological functions according to their location and receptor subtype. Multiple events are associated with the regulation of intracellular signaling by mAChRs, and a coordinated balance of the molecular mechanisms governing receptor signaling, desensitization, resensitization, and mitogenic signaling is known to occur in various cell types. Most of the actions of acetylcholine (ACh) in the male reproductive tract are induced by its effects on mAChRs, but the role of specific mAChR subtypes on male reproductive function and fertility are still not well understood. The rat efferent ductules and epididymis are androgen-dependent tissues of the male reproductive tract, with important roles in the process to form a viable and fertile sperm. In the present study, aspects of the expression, localization, and potential function of mAChR subtypes in rat efferent ductules and epididymis are reviewed. Furthermore, evidences for the implication of mAChRs in the regulation of protein synthesis and secretion in these tissues are presented. Taken together, the studies contribute to our understanding about physiological aspects of mAChR and mechanisms by which the cholinergic system affects male reproduction.

Focal adhesion kinase is a blood-testis barrier regulator.

In mammalian testes, such as rats, the mechanism(s) that regulate blood-testis barrier (BTB) restructuring at stages VIII-IX of the seminiferous epithelial cycle of spermatogenesis to facilitate the transit of preleptotene/leptotene spermatocytes is not known. This is due to the lack of information on the regulatory proteins at the BTB. Herein, focal adhesion kinase (FAK), a nonreceptor protein tyrosine kinase, is shown to structurally interact with occludin and ZO-1 to form a functional protein complex at the BTB. Its expression at the BTB in the seminiferous epithelium is stage specific, being lowest at stage VIII-IX tubules, analogous to the expression pattern of occludin. Using primary Sertoli cells cultured in vitro with an established tight junction (TJ) permeability barrier that mimics the BTB in vivo, the knockdown of FAK by RNAi led to a transient disruption of the TJ barrier. This was accompanied by a loss of association between occludin and ZO-1, likely the result of reduced occludin phosphorylation at Tyr and Ser residues, but not Thr, which in turn led to a redistribution of occludin at the Sertoli-Sertoli cell interface, moving from cell membrane into cell cytosol, thereby disrupting the BTB. These findings suggest that a similar mechanism is in place in the testis in vivo to regulate BTB restructuring to facilitate the transit of primary spermatocytes. Furthermore, FAK was shown to be a molecular target of cadmium because its knockdown would desensitize Sertoli cells to cadmium-induced TJ barrier disruption. In summary, FAK is a unique regulator of BTB dynamics in the testis.

An occludin-focal adhesion kinase protein complex at the blood-testis barrier: a study using the cadmium model.

Several integral membrane proteins that constitute the blood-testis barrier (BTB) in mammalian testes, in particular rodents, are known to date. These include tight junction (TJ) proteins (e.g. occludin, junctional adhesion molecule-A, claudins), basal ectoplasmic specialization proteins (e.g. N-cadherin), and gap junction proteins (e.g. connexin43). However, the regulators (e.g. protein kinases and phosphatases) that affect these proteins, such as their interaction with the cytoskeletal actin, which in turn confer cell adhesion at the TJ, remain largely unknown. We report herein that focal adhesion kinase (FAK) is a putative interacting partner of occludin, but not claudin-11 or junctional adhesion molecule-A. Immunohistochemistry and fluorescence microscopy studies illustrated that the expression of FAK in the seminiferous epithelium of adult rat testes was stage specific. FAK colocalized with occludin at the BTB in virtually all stages of the seminiferous epithelial cycle but considerably diminished in stages VIII-IX, at the time of BTB restructuring to facilitate the transit of primary leptotene spermatocytes. Using Sertoli cells cultured in vitro with established TJ-permeability barrier and ultrastructures of TJ, basal ectoplasmic specialization and desmosome-like junction that mimicked the BTB in vivo, FAK was shown to colocalize with occludin and zonula occludens-1 (ZO-1) at the Sertoli-Sertoli cell interface. When these Sertoli cell cultures were treated with CdCl(2) to perturb the TJ-barrier function, occludin underwent endocytic-mediated internalization in parallel with FAK and ZO-1. Thus, these findings demonstrate that FAK is an integrated regulatory component of the occludin-ZO-1 protein complex, suggesting that functional studies can be performed to study the role of FAK in BTB dynamics.

Cadmium-induced testicular injury.

Cadmium (Cd) is an environmental toxicant and an endocrine disruptor in humans and rodents. Several organs (e.g., kidney, liver) are affected by Cd and recent studies have illustrated that the testis is exceedingly sensitive to Cd toxicity. More important, Cd and other toxicants, such as heavy metals (e.g., lead, mercury) and estrogenic-based compounds (e.g., bisphenols) may account for the recent declining fertility in men among developed countries by reducing sperm count and testis function. In this review, we critically discuss recent data in the field that have demonstrated the Cd-induced toxicity to the testis is probably the result of interactions of a complex network of causes. This is likely to involve the disruption of the blood-testis barrier (BTB) via specific signal transduction pathways and signaling molecules, such as p38 mitogen-activated protein kinase (MAPK). We also summarize current studies on factors that confer and/or regulate the testis sensitivity to Cd, such as Cd transporters and metallothioneins, the impact of Cd on the testis as an endocrine disruptor and oxidative stress inducer, and how it may disrupt the Zn(2+) and/or Ca(2+) mediated cellular events. While much work is needed before a unified mechanistic pathway of Cd-induced testicular toxicity emerges, recent studies have helped to identify some of the likely mechanisms and/or events that take place during Cd-induced testis injury. Furthermore, some of the recent studies have shed lights on potential therapeutic or preventive approaches that can be developed in future studies by blocking or minimizing the destructive effects of Cd to testicular function in men.

17beta-estradiol induces the translocation of the estrogen receptors ESR1 and ESR2 to the cell membrane, MAPK3/1 phosphorylation and proliferation of cultured immature rat Sertoli cells.

The aim of the present study was to determine the mechanisms involved in estrogen actions in cultured rat Sertoli cells. RT-PCR detected transcripts for the estrogen receptors ESR1 and ESR2 in cultured immature Sertoli cells and in the testis of 15-, 28-, and 120-day-old rats. The expression of ESR1 and ESR2 was confirmed in Sertoli cells by immunofluorescence and Western blot. Immunohistochemistry with cryosections of testes from immature and adult rats revealed that ESR1 is present in Sertoli, Leydig, and some peritubular myoid cells, and ESR2 is present in multiple cell types, including germ cells. Treatment of Sertoli cells with 17beta-estradiol (E(2)) induced a translocation of ESR1 and ESR2 to the plasma membrane and a concomitant phosphorylation of MAPK3/1. Both effects reached a maximum after 10 min and were blocked by PP2, an inhibitor of the SRC family of protein tyrosine kinases, and by the antiestrogen ICI 182,780 (ICI). MAPK3/1 phosphorylation was also decreased in the presence of AG 1478, an inhibitor of the epidermal growth factor receptor (EGFR) kinase, and in the presence of MAP2K1/2 inhibitor UO126. Treatment with E(2) for 24 h increased the incorporation of [methyl-(3)H]thymidine, which was blocked by ICI. These results indicate that E(2) activates an SRC-mediated translocation of estrogen receptors to the plasma membrane, which results in the activation of EGFR and the mitogen-activated protein kinase signaling pathway. In addition, activation of ESR1 and/or ESR2 by E(2) is involved in proliferation of immature Sertoli cells. The estrogen actions in Sertoli cells might be a key step mediating cellular events important for spermatogenesis and fertility.

Expression and localization of muscarinic acetylcholine receptor subtypes in rat efferent ductules and epididymis.

The expression of muscarinic acetylcholine receptor (mAChR) subtypes (M(1)-M(5)) was studied in the rat efferent ductules and epididymis at the mRNA and protein levels. The relative abundance of each mAChR transcript subtype differed depending on the tissue and the epididymal region analyzed. The M(1) mAChR mRNA level was more abundant in the efferent ductules than in the caput and cauda of the epididymis. The M(2) mAChR mRNA level was similar between the efferent ductules and caput of the epididymis and higher in the cauda region. The M(3) mAChR mRNA level was low in the efferent ductules and caput of the epididymis, but high levels were detected in the cauda region. mRNAs for M(4) and M(5) mAChRs were not detected in these tissues. Our studies indicated a variable degree of immunostaining for each mAChR subtype in a cell-type and tissue-specific pattern. M(1) mAChR was detected over the efferent ductule epithelium. M(2) and M(3) mAChRs were observed in the apical region of the ciliated cells. Apical and narrow cells of the initial segment showed distinct staining by M(1) antibody, whereas a supranuclear reaction was noted in the principal cells of the caput of the epididymis. In addition, staining for M(1) and M(2) mAChRs was visible in the apical membrane of some epithelial cells of the cauda region. M(3) mAChR was detected in the peritubular smooth muscle of the efferent ductules and epididymis. Functional studies suggested the involvement of this subtype in epididymal tubule contraction. Thus, the cell-specific expression of the various mAChR subtypes in the efferent ductules and epididymis suggests that these receptors play a role in the modulation of luminal fluid composition and smooth muscle contraction.