Integrating human health and environmental health into the DPSIR framework: a tool to identify research opportunities for sustainable and healthy communities.

The U.S. Environmental Protection Agency has recently realigned its research enterprise around the concept of sustainability. Scientists from across multiple disciplines have a role to play in contributing the information, methods, and tools needed to more fully understand the long-term impacts of decisions on the social and economic sustainability of communities. Success will depend on a shift in thinking to integrate, organize, and prioritize research within a systems context. We used the Driving forces-Pressures-State-Impact-Response (DPSIR) framework as a basis for integrating social, cultural, and economic aspects of environmental and human health into a single framework. To make the framework broadly applicable to sustainability research planning, we provide a hierarchical system of DPSIR keywords and guidelines for use as a communication tool. The applicability of the integrated framework was first tested on a public health issue (asthma disparities) for purposes of discussion. We then applied the framework at a science planning meeting to identify opportunities for sustainable and healthy communities research. We conclude that an integrated systems framework has many potential roles in science planning, including identifying key issues, visualizing interactions within the system, identifying research gaps, organizing information, developing computational models, and identifying indicators.

Roles of reactive oxygen species and antioxidants in ovarian toxicity.

Proper functioning of the ovary is critical to maintain fertility and overall health, and ovarian function depends on the maintenance and normal development of ovarian follicles. This review presents evidence about the potential impact of oxidative stress on the well-being of primordial, growing and preovulatory follicles, as well as oocytes and early embryos, examining cell types and molecular targets. Limited data from genetically modified mouse models suggest that several antioxidant enzymes that protect cells from reactive oxygen species (ROS) may play important roles in follicular development and/or survival. Exposures to agents known to cause oxidative stress, such as gamma irradiation, chemotherapeutic drugs, or polycyclic aromatic hydrocarbons, induce rapid primordial follicle loss; however, the mechanistic role of ROS has received limited attention. In contrast, ROS may play an important role in the initiation of apoptosis in antral follicles. Depletion of glutathione leads to atresia of antral follicles in vivo and apoptosis of granulosa cells in cultured antral follicles. Chemicals, such as cyclophosphamide, dimethylbenzanthracene, and methoxychlor, increase proapoptotic signals, preceded by increased ROS and signs of oxidative stress, and cotreatment with antioxidants is protective. In oocytes, glutathione levels change rapidly during progression of meiosis and early embryonic development, and high oocyte glutathione at the time of fertilization is required for male pronucleus formation and for embryonic development to the blastocyst stage. Because current evidence suggests that oxidative stress can have significant negative impacts on female fertility and gamete health, dietary or pharmacological intervention may prove to be effective strategies to protect female fertility.

Preliminary examination of polymorphisms of GSTM1, GSTT1, and GSTZ1 in relation to semen quality.

BACKGROUND: Environmental, lifestyle, and occupational exposures on semen quality have been investigated in epidemiological studies with inconsistent results. Genetic factors involved in toxicant activation and detoxification have been examined in relation to the risk of outcomes such as cancer, cardiovascular, and neurologic disorders. However, the effect of common genetic variants in the metabolism of toxicants on semen quality parameters has rarely been evaluated. In this analysis, we evaluated functional SNPs of three genes of the glutathione-S-transferase (GSTM1, GSTT1, GSTZ1) enzyme family. METHODS: Participants were 228 presumed fertile men recruited as part of a community-based study. Semen outcome data from this study included total sperm count and concentration, sperm morphology, and sperm DNA integrity and chromatin maturity. DNA was obtained from 162 men from a mouth-rinse sample and genotyped for the presence of GSTT1-1 and GSTM1-1 null genotypes and the GSTZ1 SNPs at positions 94 (rs3177427) and 124 (rs3177429). We used multivariable linear regression to assess the relationship between each genotype and sperm outcomes. RESULTS: Overall, our results did not reveal a consistent pattern between GSTM1 and GSTZ genotypes and increased occurrence of adverse sperm outcomes. However, the GSTT1 non-null genotype yielded the coefficients with the largest magnitude for sperm count and sperm concentration (beta=-0.528, 95% CI -1.238 to 0.199 and beta=-0.353, 95% CI -0.708 to 0.001, respectively), suggesting that it might be adverse. CONCLUSIONS: These results indicate that common polymorphisms in GST genes do not negatively impact sperm parameters in healthy men with good semen quality. Contrary to expectations, the GSTT1 non-null genotype was associated with reduced sperm concentration and count in semen. Further study with a larger study size and inclusion of gene-exposure interactions is warranted.

Sperm DNA: organization, protection and vulnerability: from basic science to clinical applications--a position report.

This article reports the results of the most recent in a series of EHSRE workshops designed to synthesize the current state of the field in Andrology and provide recommendations for future work (for details see Appendix). Its focus is on methods for detecting sperm DNA damage and potential application of new knowledge about sperm chromatin organization, vulnerability and repair to improve the diagnosis and treatment of clinical infertility associated with that damage. Equally important is the use and reliability of these tests to identify the extent to which environmental contaminants or pharmaceutical agents may contribute to the incidence of sperm DNA damage and male fertility problems. A working group (for workshop details, see Appendix) under the auspices of ESHRE met in May 2009 to assess the current knowledgebase and suggest future basic and clinical research directions. This document presents a synthesis of the working group's understanding of the recent literature and collective discussions on the current state of knowledge of sperm chromatin structure and function during fertilization. It highlights the biological, assay and clinical uncertainties that require further research and ends with a series of 5 key recommendations.

The effect of ambient air pollution on sperm quality.

BACKGROUND: Research has suggested an association with ambient air pollution and sperm quality. OBJECTIVES: We investigated the effect of exposure to ozone (O3) and particulate matter < 2.5 microm in aerodynamic diameter (PM2.5) on sperm quality. METHODS: We reexamined a previous cohort study of water disinfection by-products to evaluate sperm quality in 228 presumed fertile men with different air pollution profiles. Outcomes included sperm concentration, total sperm per ejaculate (count), and morphology, as well as DNA integrity and chromatin maturity. Exposures to O3 and PM2.5 were evaluated for the 90-day period before sampling. We used multivariable linear regression, which included different levels of adjustment (i.e., without and with season and temperature) to assess the relationship between exposure to air pollutants during key periods of sperm development and adverse sperm outcomes. RESULTS: Sperm concentration and count were not associated with exposure to PM2.5, but there was evidence of an association (but not statistically significant) with O3 concentration and decreased sperm concentration and count. Additionally, a significant increase in the percentage of sperm cells with cytoplasmic drop [beta = 2.64; 95% confidence interval (CI), 0.215.06] and abnormal head (beta = 0.47; 95% CI, 0.030.92) was associated with PM2.5 concentration in the base model. However, these associations, along with all other sperm outcomes, were not significantly associated with either pollutant after controlling for season and temperature. Overall, although we found both protective and adverse effects, there was generally no consistent pattern of increased abnormal sperm quality with elevated exposure to O3 or PM2.5. CONCLUSIONS: Exposures to O3 or PM2.5 at levels below the current National Ambient Air Quality Standards were not associated with statistically significant decrements in sperm outcomes in this cohort of fertile men. However, some results suggested effects on sperm concentration, count, and morphology.

GSTM1 genotype influences the susceptibility of men to sperm DNA damage associated with exposure to air pollution.

Previous studies have provided evidence for an association between exposure to high levels of air pollution and increased DNA damage in human sperm. In these studies DNA damage was measured using the sperm chromatin structure assay (SCSA) wherein the percentage of sperm with abnormal chromatin/fragmented DNA is determined and expressed as % DNA fragmentation index (%DFI). Here we extend these observations to address the following hypothesis: men who are homozygous null for glutathione-S-transferase M1 (GSTM1-) are less able to detoxify reactive metabolites of carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) found in air pollution. Consequently they are more susceptible to the effects of air pollution on sperm chromatin. Using a longitudinal study design in which men provided semen samples during periods of both low (baseline) and episodically high air pollution, this study revealed a statistically significant association between GSTM1 null genotype and increased SCSA-defined %DFI (beta=0.309; 95% CI: 0.129, 0.489). Furthermore, GSTM1 null men also showed higher %DFI in response to exposure to intermittent air pollution (beta=0.487; 95% CI: 0.243, 0.731). This study thus provides novel evidence for a gene-environment interaction between GSTM1 and air pollution (presumably c-PAHs). The significance of the findings in this study with respect to fertility status is unknown. However, it is biologically plausible that increases in %DFI induced by such exposures could impact the risk of male sub/infertility, especially in men who naturally exhibit high levels of %DFI.

The healthy men study: an evaluation of exposure to disinfection by-products in tap water and sperm quality.

BACKGROUND: Chlorination of drinking water generates disinfection by-products (DBPs), which have been shown to disrupt spermatogenesis in rodents at high doses, suggesting that DBPs could pose a reproductive risk to men. In this study we assessed DBP exposure and testicular toxicity, as evidenced by altered semen quality. METHODS: We conducted a cohort study to evaluate semen quality in men with well-characterized exposures to DBPs. Participants were 228 presumed fertile men with different DBP profiles. They completed a telephone interview about demographics, health history, water consumption, and other exposures and provided a semen sample. Semen outcomes included sperm concentration and morphology, as well as DNA integrity and chromatin maturity. Exposures to DBPs were evaluated by incorporating data on water consumption and bathing and showering with concentrations measured in tap water. We used multivariable linear regression to assess the relationship between exposure to DBPs and adverse sperm outcomes. RESULTS: The mean (median) sperm concentration and sperm count were 114.2 (90.5) million/mL and 362 (265) million, respectively. The mean (median) of the four trihalomethane species (THM4) exposure was 45.7 (65.3) microg/L, and the mean (median) of the nine haloacetic acid species (HAA9) exposure was 30.7 (44.2) microg/L. These sperm parameters were not associated with exposure to these classes of DBPs. For other sperm outcomes, we found no consistent pattern of increased abnormal semen quality with elevated exposure to trihalomethanes (THMs) or haloacetic acids (HAAs). The use of alternate methods for assessing exposure to DBPs and site-specific analyses did not change these results. CONCLUSIONS: The results of this study do not support an association between exposure to levels of DBPs near or below regulatory limits and adverse sperm outcomes in humans.

Assessing human germ-cell mutagenesis in the Postgenome Era: a celebration of the legacy of William Lawson (Bill) Russell.

Birth defects, de novo genetic diseases, and chromosomal abnormality syndromes occur in approximately 5% of all live births, and affected children suffer from a broad range of lifelong health consequences. Despite the social and medical impact of these defects, and the 8 decades of research in animal systems that have identified numerous germ-cell mutagens, no human germ-cell mutagen has been confirmed to date. There is now a growing consensus that the inability to detect human germ-cell mutagens is due to technological limitations in the detection of random mutations rather than biological differences between animal and human susceptibility. A multidisciplinary workshop responding to this challenge convened at The Jackson Laboratory in Bar Harbor, Maine. The purpose of the workshop was to assess the applicability of an emerging repertoire of genomic technologies to studies of human germ-cell mutagenesis. Workshop participants recommended large-scale human germ-cell mutation studies be conducted using samples from donors with high-dose exposures, such as cancer survivors. Within this high-risk cohort, parents and children could be evaluated for heritable changes in (a) DNA sequence and chromosomal structure, (b) repeat sequences and minisatellites, and (c) global gene expression profiles and pathways. Participants also advocated the establishment of a bio-bank of human tissue samples from donors with well-characterized exposure, including medical and reproductive histories. This mutational resource could support large-scale, multiple-endpoint studies. Additional studies could involve the examination of transgenerational effects associated with changes in imprinting and methylation patterns, nucleotide repeats, and mitochondrial DNA mutations. The further development of animal models and the integration of these with human studies are necessary to provide molecular insights into the mechanisms of germ-cell mutations and to identify prevention strategies. Furthermore, scientific specialty groups should be convened to review and prioritize the evidence for germ-cell mutagenicity from common environmental, occupational, medical, and lifestyle exposures. Workshop attendees agreed on the need for a full-scale assault to address key fundamental questions in human germ-cell environmental mutagenesis. These include, but are not limited to, the following: Do human germ-cell mutagens exist? What are the risks to future generations? Are some parents at higher risk than others for acquiring and transmitting germ-cell mutations? Obtaining answers to these, and other critical questions, will require strong support from relevant funding agencies, in addition to the engagement of scientists outside the fields of genomics and germ-cell mutagenesis.

The healthy men study: design and recruitment considerations for environmental epidemiologic studies in male reproductive health.

OBJECTIVE: To describe study design, conduct and response, and participant characteristics. DESIGN: Prospective cohort study. SETTING: Participants were male partners of women who were enrolled in a community-based prospective cohort study of drinking water disinfection by-products and pregnancy health. PATIENT(S): Two hundred thirty presumed fertile men recruited from 3 study sites in the United States. INTERVENTION(S): Men completed a telephone interview about demographics, health history, and exposures and provided a semen sample that was express mailed to the study laboratory. MAIN OUTCOME MEASURE(S): Response and participation rates, participant demographics, and lifestyle exposures. RESULT(S): We obtained a high participation rate (84%) among men who were located, but a low overall response rate (25%). Participants were more likely to be white, more highly educated, be married, and have a higher household income than the underlying study cohort. CONCLUSION(S): Our multisite study design may be applicable to the study of community environmental factors and reproductive health of men. Our design was efficient in that men from geographically disparate sites could be recruited, a semen sample was collected at home, and a telephone interview was conducted from a central study site. Despite these design features, the low response rates may suggest selection bias that can be addressed partially in the analysis.

Workgroup report: Implementing a national occupational reproductive research agenda--decade one and beyond.

The initial goal of occupational reproductive health research is to effectively study the many toxicants, physical agents, and biomechanical and psychosocial stressors that may constitute reproductive hazards in the workplace. Although the main objective of occupational reproductive researchers and clinicians is to prevent recognized adverse reproductive outcomes, research has expanded to include a broader spectrum of chronic health outcomes potentially affected by reproductive toxicants. To aid in achieving these goals, the National Institute for Occupational Safety and Health, along with its university, federal, industry, and labor colleagues, formed the National Occupational Research Agenda (NORA) in 1996. NORA resulted in 21 research teams, including the Reproductive Health Research Team (RHRT). In this report, we describe progress made in the last decade by the RHRT and by others in this field, including prioritizing reproductive toxicants for further study; facilitating collaboration among epidemiologists, biologists, and toxicologists; promoting quality exposure assessment in field studies and surveillance; and encouraging the design and conduct of priority occupational reproductive studies. We also describe new tools for screening reproductive toxicants and for analyzing mode of action. We recommend considering outcomes such as menopause and latent adverse effects for further study, as well as including exposures such as shift work and nanomaterials. We describe a broad domain of scholarship activities where a cohesive system of organized and aligned work activities integrates 10 years of team efforts and provides guidance for future research.

Episodic air pollution is associated with increased DNA fragmentation in human sperm without other changes in semen quality.

BACKGROUND: This study examined potential associations between exposure to episodes of air pollution and alterations in semen quality. The air pollution, resulting from combustion of coal for industry and home heating in the Teplice district of the Czech Republic, was much higher during the winter than at other times of year with peaks exceeding US air quality standards. METHODS: Young men from Teplice were sampled up to seven times over 2 years allowing evaluation of semen quality after periods of exposure to both low and high air pollution. Routine semen analysis (sperm concentration, motility and morphology) and tests for sperm aneuploidy and chromatin integrity were performed, comparing measurements within each subject. Exposure was classified as high or low based on data from ambient air pollution monitoring. RESULTS: Using repeated measures analysis, a significant association was found between exposure to periods of high air pollution (at or above the upper limit of US air quality standards) and the percentage of sperm with DNA fragmentation according to sperm chromatin structure assay (SCSA). Other semen measures were not associated with air pollution. CONCLUSION: Exposure to intermittent air pollution may result in sperm DNA damage and thereby increase the rates of male-mediated infertility, miscarriage, and other adverse reproductive outcomes.

Acute exposure to molinate alters neuroendocrine control of ovulation in the rat.

Molinate, a thiocarbamate herbicide, has been reported to impair reproductive capability in the male rat and alter pregnancy outcome in a two-generation study. Published data are lacking on the effects of acute exposure to molinate in the female. Based on this work and our previous observations with related dithiocarbamate compounds, we hypothesized that a single exposure to molinate during the critical window for the neural trigger of ovulation on the day of proestrus (PRO) would block the luteinizing hormone (LH) surge and delay ovulation. To examine the effect of molinate on the LH surge, ovariectomized (OVX) rats were implanted with Silastic capsules containing estradiol benzoate to mimic physiological levels on proestrus. Doses of 25 and 50 mg/kg molinate significantly suppressed LH and prolactin secretion. Intact regularly cycling females gavaged with 0, 25, or 50 mg/kg molinate at 1300 h on PRO were examined on estrus or estrus +1 day for the presence of oocytes in the oviduct. All control females had oocytes in the oviduct on estrus. Molinate doses of 6.25 to 50 mg/kg delayed ovulation for 24 h. Estrous cyclicity was examined after daily exposure to 50 mg/kg (21 days). Estrous cyclicity was irregular in the molinate group, showing extended days in estrus. Two experiments were conducted to determine whether molinate blocked the LH surge via a central nervous system (CNS) mode of action or via an alteration in pituitary response. In the first experiment, we evaluated the release of LH in control and molinate-treated rats after a bolus dose of exogenous GnRH. Luteinizing hormone release was comparable in the two groups, suggesting that the effect of molinate is centrally mediated. To further examine the potential role of the CNS, we examined the pulsatile release of LH present in the long-term OVX females. In this model, the pulsatile pattern of LH secretion is directly correlated with GnRH release from the hypothalamus. A significant decrease in the LH pulse frequency was observed in molinate-treated females. These results indicate that molinate is able to delay ovulation by suppressing the LH surge on the day of proestrus and that the brain is the primary target site for the effects on pituitary hormone secretion.

Glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme, is required for sperm motility and male fertility.

Although glycolysis is highly conserved, it is remarkable that several unique isozymes in this central metabolic pathway are found in mammalian sperm. Glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS) is the product of a mouse gene expressed only during spermatogenesis and, like its human ortholog (GAPD2), is the sole GAPDH isozyme in sperm. It is tightly bound to the fibrous sheath, a cytoskeletal structure that extends most of the length of the sperm flagellum. We disrupted Gapds expression by gene targeting to selectively block sperm glycolysis and assess its relative importance for in vivo sperm function. Gapds(-/-) males were infertile and had profound defects in sperm motility, exhibiting sluggish movement without forward progression. Although mitochondrial oxygen consumption was unchanged, sperm from Gapds(-/-) mice had ATP levels that were only 10.4% of those in sperm from WT mice. These results imply that most of the energy required for sperm motility is generated by glycolysis rather than oxidative phosphorylation. Furthermore, the critical role of glycolysis in sperm and its dependence on this sperm-specific enzyme suggest that GAPDS is a potential contraceptive target, and that mutations or environmental agents that disrupt its activity could lead to male infertility.

The value of home-based collection of biospecimens in reproductive epidemiology.

Detection, quantification, and prognosis of environmental exposures in humans has been vastly enhanced by the ability of epidemiologists to collect biospecimens for toxicologic or other laboratory evaluation. Ease of collection and level of invasiveness are commonly cited reasons why study participants fail to provide biospecimens for research purposes. The use of methodologies for the collection of biospecimens in the home offers promise for improving the validity of health effects linked to environmental exposures while maximizing the number and type of specimens capable of being collected in a timely and cost-effective manner. In this review we examine biospecimens (urine and blood) that have been successfully collected from the home environment. Related issues such as storage and transportation will also be examined as well as promising new approaches for collecting less frequently studied biospecimens (including hair follicles, breast milk, semen, and others). Such biospecimens are useful in the monitoring of reproductive development and function.

Evaluation of chromosome breakage and DNA integrity in sperm: an investigation of remote semen collection conditions.

Collection of ejaculated semen at a remote site (outside of the laboratory) would facilitate participation rates and geographic diversity in reproductive epidemiology studies. Our study addressed concerns that remote collection and overnight mail return might induce chromosome/DNA damage. We collected semen from 10 healthy men. Part of each sample was snap frozen in liquid nitrogen and the rest held at 22 +/- 1 degrees C for 24 hours in a transport container (simulating ambient temperature during overnight return) then snap frozen. DNA breakage and fragmentation were measured using tandem-label sperm-fluorescence in situ hybridization (FISH), terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), and neutral comet assay. Tandem-label sperm-FISH and TUNEL detected no statistically significant difference between sperm fresh frozen (FF) and those frozen after 24 hours (F24). The mean frequency of chromosome breakage per 10 000 cells scored in sperm-FISH for FF and F24 was 10.5 +/- 1.3 breaks and 11.2 +/- 1.1 breaks, respectively (P =.69, Student's t test). The mean frequency of TUNEL-positive cells per 2000 cells scored in FF and F24 was 136 +/- 29 and 213 +/- 28 cells, respectively, which approached but did not reach statistical significance (P = 0.07, Student's t test). The neutral comet assay detected a statistically significant difference in DNA strand breakage between the 2 groups (percentage of DNA in the tail P = 0.037; tail moment P = 0.006; and tail length P = 0.033, all Student's t test). The mean frequency of damage denoted by tail length in micro m per 100 cells scored in FF and F24 was 175.0 +/- 15.5 and 152.2 +/- 17.6 micro m, respectively. Tandem-label sperm-FISH, TUNEL, and neutral comet assay are useful analytical techniques for laboratory-based studies of human sperm genomic integrity; however, for field studies incorporating the nonrefrigerated return of semen after 24 hours, only chromosome breakage at a level that can be detected using tandem-label sperm-FISH was unaffected. TUNEL and neutral comet assay need further study before they are used in specimens collected at remote sites and transported to a central laboratory.

An occupational reproductive research agenda for the third millennium.

There is a significant public health concern about the potential effects of occupational exposure to toxic substances on reproductive outcomes. Several toxicants with reported reproductive and developmental effects are still in regular commercial or therapeutic use and thus present potential exposure to workers. Examples of these include heavy metals, organic solvents, pesticides and herbicides, and sterilants, anesthetic gases, and anticancer drugs used in health care. Many other substances are suspected of producing reproductive or developmental toxicity but lack sufficient data. Progress has been limited in identifying hazards and quantifying their potencies and in separating the contribution of these hazards from other etiologic factors. Identifying the causative agents, mechanisms by which they act, and any potential target populations, present the opportunity to intervene and protect the reproductive health of workers. The pace of laboratory studies to identify hazards and to underpin the biologic plausibility of effects in humans has not matched the pace at which new chemicals are introduced into commerce. Though many research challenges exist today, recent technologic and methodologic advances have been made that allow researchers to overcome some of these obstacles. The objective of this article is to recommend future directions in occupational reproductive health research. By bridging interdisciplinary gaps, the scientific community can work together to improve health and reduce adverse outcomes.

Abnormal fertilization is responsible for reduced fecundity following thiram-induced ovulatory delay in the rat.

Brief exposure to some pesticides, applied during a sensitive window for the neural regulation of ovulation, will block the preovulatory surge of LH and, thus, delay ovulation. Previously, we have shown that a single i.p. injection of 50 mg/kg of thiram, a dithiocarbamate fungicide that decreases norepinephrine synthesis, on proestrus (1300 h) suppresses the LH surge and delays ovulation for 24 h without altering the number of oocytes released. However, when bred, the treated dams had a decreased litter size and increased postimplantation loss. We hypothesized that the reduced litter size in thiram-delayed rats was a consequence of altered oocyte function arising from intrafollicular oocyte aging. To test this hypothesis, we examined delayed oocytes, zygotes, and 2-cell embryos for evidence of fertilization and polyspermy. In addition, we used confocal laser-scanning microscopy to evaluate and characterize cortical granule localization in oocytes and release in zygotes, because the cortical granule response is a major factor in the normal block to polyspermy. Our results demonstrate that a thiram-induced, 24-h delay in ovulation alters the fertilizability of the released oocyte. Although no apparent morphological differences were observed in the unfertilized mature oocytes released following the thiram-induced delay, the changes observed following breeding include a significant decrease in the percentage of fertilized oocytes, a significant increase in polyspermic zygotes (21%), and a 10-fold increase in the number of supernumerary sperm in the perivitelline space. Importantly, all the polyspermic zygotes exhibited an abnormal pattern of cortical granule exudate, suggestive of a relationship between abnormal cortical reaction and the polyspermy in the delayed zygotes. Because polyspermy is associated with polyploidy, abnormal development, and early embryonic death, the observed polyspermy could explain the abnormal development and decreased litter size that we observed previously following thiram-delayed ovulation.