Endocrine disruptors: Challenges and future directions in epidemiologic research.

Public concern about the impact of endocrine disrupting chemicals (EDCs) on both humans and the environment is growing steadily. Epidemiologic research provides key information towards our understanding of the relationship between environmental exposures like EDCs and human health outcomes. Intended for researchers in disciplines complementary to epidemiology, this paper highlights the importance and challenges of epidemiologic research in order to present the key elements pertaining to the design and interpretation of an epidemiologic study on EDCs. The conduct of observational studies on EDCs derives from a thoughtful research question, which will help determine the subsequent methodological choices surrounding the careful selection of the study population (including the comparison group), the adequate ascertainment of exposure(s) and outcome(s) of interest, and the application of methodological and statistical concepts more specific to epidemiology. The interpretation of epidemiologic results may be arduous due to the latency occurring between EDC exposure and certain outcome(s), the complexity in capturing EDC exposure(s), and traditional methodological and statistical issues that also deserve consideration (e.g., confounding, effect modification, non-monotonic responses). Moving forward, we strongly advocate for an integrative approach of expertise in the fields of epidemiology, exposure science, risk assessment and toxicology to adequately study the health risks associated with EDCs while tackling their challenges.

An approach to classifying occupational exposures to endocrine disrupting chemicals by sex hormone function using an expert judgment process.

Endocrine disrupting chemicals (EDCs) are exogenous substances that interfere with the endocrine system and cause adverse effects. We aimed to classify the effects of 24 known EDCs, prevalent in certain occupations, according to four modes of action (estrogenic, antiestrogenic, androgenic, and/or antiandrogenic). A literature search, stratified into four types of literature was conducted (namely: national and international agency reports; review articles; primary studies; ToxCastTM). The state of the evidence of each EDC on sex hormone function was summarized and reviewed by an expert panel. For each mode of action, the experts evaluated the likelihood of endocrine disruption in five categories: "No", "Unlikely", "Possibly", "Probably", and "Yes". Seven agents were categorized as "Yes," or having strong evidence for their effects on sex hormone function (antiandrogenic: lead, arsenic, butylbenzyl phthalate, dibutyl phthalate, dicyclohexyl phthalate; estrogenic: nonylphenol, bisphenol A). Nine agents were categorized as "Probable," or having probable evidence (antiandrogenic: bis(2-ethylhexyl)phthalate, nonylphenol, toluene, bisphenol A, diisononyl phthalate; androgenic: cadmium; estrogenic: copper, cadmium and; anti-estrogenic: lead). Two agents (arsenic, polychlorinated biphenyls) had opposing conclusions supporting both "probably" estrogenic and antiestrogenic effects. This synthesis will allow researchers to evaluate the health effects of selected EDCs with an added level of precision related to the mode of action.

Influence of Polishing Systems on Surface Roughness of Composite Resins: Polishability of Composite Resins.

OBJECTIVES: The objective of this in vitro study was to compare, with a threshold value of 200 nm, the surface roughness obtained when using 12 different polishing systems on four different composite resins (microfill, nanofill, and two nanohybrids). METHODS AND MATERIALS: A total of 384 convex specimens were made using Durafill VS, Filtek Supreme Ultra, Grandio SO, and Venus Pearl. After sandblasting and finishing with a medium-grit finishing disc, initial surface roughness was measured using a surface roughness tester. Specimens were polished using 12 different polishing systems: Astropol, HiLuster Plus, D♦Fine, Diacomp, ET Illustra, Sof-Lex Wheels, Sof-Lex XT discs, Super-Snap, Enhance/Pogo, Optrapol, OneGloss and ComposiPro Brush (n=8). The final surface roughness was measured, and data were analyzed using two-way analysis of variance. Pairwise comparisons were made using protected Fisher least significant difference. RESULTS: There were statistical differences in the final surface roughness between polishing systems and between composite resins (p<0.05). The highest surface roughness was observed for all composite resins polished with OneGloss and ComposiPro Brush. Enhance/Pogo and Sof-Lex Wheels produced a mean surface roughness greater than the 200-nm threshold on Filtek Supreme Ultra, Grandio SO, and Venus Pearl. Data showed that there was an interaction between the composite resins and the polishing systems. CONCLUSIONS: A single polishing system does not perform equally with all composite resins. Except for Optrapol, multi-step polishing systems performed generally better than one-step systems. Excluding Enhance/Pogo, diamond-impregnated polishers led to lower surface roughness. Durafill VS, a microfill composite resin, may be polished more predictably with different polishers.

Insidious weight gain in prepubertal seized rats treated with an atypical neuroleptic: the role of food consumption, fluid consumption, and spontaneous ambulatory activity.

Extreme obesity slowly develops in female rats over the months following seizures induced by a single systemic injection of lithium and pilocarpine if the resulting limbic seizures are treated with the atypical neuroleptic acepromazine (but not with ketamine). To discern the contributions from food consumption, water consumption, and (daytime and nighttime) activity to this weight gain, these behaviors were monitored for 4 months, about 2 months after seizure induction. The results indicated that the rats that underwent the obesity procedure exhibited 50% heavier body weights and consumed 42% more food than the reference group, which included rats that had been induced to seize but treated with ketamine. There were no statistically significant differences between groups with respect to either water consumption or (daytime or nighttime) activity. Factor analyses of data for individual rats verified the dissociation between activity and weight gain for the obese rats. The results suggest that the progressive weight gains are centrally mediated and are not secondary to diminished activity or altered fluid consumption.

Behavioral changes in adult rats after prenatal exposures to complex, weak magnetic fields.

More than 100 adult male and female albino Wistar rats that had been exposed during their entire prenatal development to one of two patterns of magnetic fields and one of four intensities (reference: 5 to 20 nT; low: 30 to 50 nT; medium: 90 to 580 nT; high: 590 nT to 1.2 microT) were tested for their capacity for two forms of classical conditioning. The rats exposed for 10 sec every 50 sec to a field composed of successive 200 msec sequences of several different patterns known to produce physiological effects exhibited significantly more intense conditioned fear and taste aversion than those exposed continuously to a single frequency-modulated pattern. The behavioral differences, relative to the reference group ("controls"), were greatest for rats exposed to the 30 to 50 nT or 90 to 580 nT (low to medium intensities) for both patterns of fields. These results suggest that prenatal exposure to physiologically-patterned magnetic fields within a specific "window" of intensities that overlap with values found in many human habitats may produce long-term changes in behaviors.

Common evolution of waprin and kunitz-like toxin families in Australian venomous snakes.

The venoms of Australian snakes contain a myriad of pharmacologically active toxin components. This study describes the identification and comparative analysis of two distinct toxin families, the kunitztype serine protease inhibitors and waprins, and demonstrates a previously unknown evolutionary link between the two. Multiple cDNA and full-length gene isoforms were cloned and shown to be composed of three exons separated by two introns. A high degree of identity was observed solely within the first exon which coded for the propeptide sequence and its cleavage site, and indicates that each toxin family has arisen from a gene duplication event followed by diversification only within the portion of the gene coding for the functional toxin. It is proposed that while the mechanism of toxin secretion is highly conserved, diversification of mature toxin sequences allows for the existence of multiple protein isoforms in the venom to adapt to variations within the prey environment.

Altered blood chemistry and hippocampal histomorphology in adult rats following prenatal exposure to physiologically-patterned, weak (50-500 nanoTesla range) magnetic fields.

PURPOSE: To discern changes in blood chemistry, cerebral sizes, and hippocampal cytomorphology in adult male and female albino Wistar rats that had been exposed during their entire prenatal development to one of two patterns of magnetic fields and one of four intensities: Very low 5 - 20 nT; low 30 - 50 nT; medium 90 - 580 nT; and high 590 nT to 1.2 microT. MATERIALS AND METHODS: A total of 48 pregnant females were exposed to either a repetitive frequency-modulated magnetic field or to a complex sequence of 50, 200-msec physiologically-patterned fields. As adults blood, cerebral, and histomorphological data were obtained from the 137 rats that had been exposed to one of these eight conditions. RESULTS: Compared to other groups, adult rats that had been exposed prenatally to the physiologically-patterned magnetic fields at the low (30 - 50 nT) and medium (90 - 580 nT) intensities exhibited peak elevations of aminotransaminase, glucose, and uric acid. Numbers of cytometric anomalies were also significantly elevated within regions of the hippocampus known for neuronal neogenesis in adults. CONCLUSIONS: The results suggest that a common factor in cellular adhesion or plasticity might be permanently altered by prenatal exposure to a narrow intensity of a series of physiologically-patterned magnetic fields.

Distinct activities of novel neurotoxins from Australian venomous snakes for nicotinic acetylcholine receptors.

Envenomation from Australian elapid snakes results in a myriad of neurological effects due to post-synaptic neurotoxins that bind and inhibit nicotinic acetylcholine receptors (nAChRs) of neurons and muscle fibres. However, despite the significant physiological effects of these toxins, they have remained largely undercharacterised at the molecular level. This study describes the identification and comparative analysis of multiple neurotoxin isoforms from ten Australian snakes, including functional characterisation of two of these isoforms, Os SNTX-1 from Oxyuranus scutellatus and the more potent Pt LNTX-1 from Pseudonaja textilis. Electrophysiological recordings from adrenal chromaffin cells demonstrate that both neurotoxins act as competitive antagonists of nAChRs in a concentration-dependent manner. Their effects upon spontaneous and nerve-evoked membrane responses at the amphibian neuromuscular junction provide further evidence that both toxins bind muscle nAChRs in an irreversible manner. This study represents one of the most comprehensive descriptions to date of the sequences and activity of individual Australian elapid neurotoxins.

Enhanced mortality of rat pups following inductions of epileptic seizures after perinatal exposures to 5 nT, 7 Hz magnetic fields.

While investigating the effects of weak complex magnetic fields upon neuroplasticity following induction of early epilepsy, an unprecedented increase in post-seizure mortality (76%) was observed for young rats that had been exposed perinatally to 7 Hz magnetic fields with maximum intensities around 5 nT. Pups exposed to less intense or more intense fields of this frequency did not display this magnitude of significant mortality. Perinatal exposure through the maternal water supply to either a putative nitric oxide donor or inhibitor did not affect this mortality. The non-linear relationship between perinatal 7 Hz magnetic field intensity and post-seizure mortality may be considered analogous to the non-linear relationship between the molarity of ligands and their sequestering to receptor subtypes. These unexpected results suggest that exposure to apparently innocuous stimuli during early development may affect vulnerability to stimuli presented later in ontogeny.

Ambulatory effects of brief exposures to magnetic fields changing orthogonally in space over time.

In a within-subject design adult male rats were exposed for 15 min once per day or night to one of two patterns of complex magnetic fields (0.5 to 1 micro T) rotated in space once every 2 s or 20 s through each of the three spatial dimensions and then simultaneously through all three dimensions. Open field behavior was then measured for ambulation, defecation, and grooming. The rats displayed about twice the ambulation after when the fields had been present compared to when they had not. The burst-firing field elicited the greatest ambulation when presented during the night whereas the frequency-modulated pattern elicited the greatest ambulation when presented during the day. These results suggest that robust behavioral changes can occur when rats are exposed for 15 min to complex spatiotemporal configurations of weak magnetic fields.

Experimental facilitation of the sensed presence is predicted by the specific patterns of the applied magnetic fields, not by suggestibility: re-analyses of 19 experiments.

If all experiences are generated by brain activity, then experiences of God and spirits should also be produced by the appropriate cerebral stimulation. During the last 15 years experiments have shown that the sensed presence of a "Sentient Being" can be reliably evoked by very specific temporal patterns of weak (<1 microT) transcerebral magnetic fields applied across the temporoparietal region of the two hemispheres. Recently Granqvist et al. (2005) attributed these effects to suggestibility and exotic beliefs. Re-analyses with additional data for 407 subjects (19 experiments) showed that the magnetic configurations, not the subjects' exotic beliefs or suggestibility, were responsible for the experimental facilitation of sensing a presence. On the other hand, the subjects' histories of sensed presences before exposure to the experimental setting were moderately correlated with exotic beliefs and temporal lobe sensitivity. Several recent experiments have shown that the side attributed to the presence at the time of the experience is sensitive to the temporal parameters of the fields, the hemisphere to which they are maximized, and the person's a priori beliefs. The importance of verifying the specific timing and temporal pattern of the software-generated fields and following an effective protocol is emphasized.

Identification and analysis of venom gland-specific genes from the coastal taipan (Oxyuranus scutellatus) and related species.

Australian terrestrial elapid snakes contain amongst the most potently toxic venoms known. However, despite the well-documented clinical effects of snake bite, little research has focussed on individual venom components at the molecular level. To further characterise the components of Australian elapid venoms, a complementary (cDNA) microarray was produced from the venom gland of the coastal taipan (Oxyuranus scutellatus) and subsequently screened for venom gland-specific transcripts. A number of putative toxin genes were identified, including neurotoxins, phospholipases, a pseudechetoxin-like gene, a venom natriuretic peptide and a nerve growth factor together with other genes involved in cellular maintenance. Venom gland-specific components also included a calglandulin-like protein implicated in the secretion of toxins from the gland into the venom. These toxin transcripts were subsequently identified in seven other related snake species, producing a detailed comparative analysis at the cDNA and protein levels. This study represents the most detailed description to date of the cloning and characterisation of different genes associated with envenomation from Australian snakes.

Extreme obesity in female rats following prepuberal induction of lithium-pilocarpine seizures and a single injection of acepromazine.

Seizures were induced in female Wistar albino rats at either 35 or 55 days of age with a single systemic injection of lithium (3 mEq/kg) and pilocarpine (30 mg/kg); the rats were then treated with the atypical neuroleptic acepromazine (25 mg/kg). These rats manifested progressive weight gain for the rest of their lives. The effect was conspicuous by casual observation 6 weeks after treatment and occurred primarily in those rats that later developed spontaneous seizures. After 1 year, the rats were obese (>1000 g). Such weight gains, associated with almost three times the serum triglyceride levels, were not observed in male rats and have not been observed in hundreds of female rats that received this treatment as adults. Single postseizure injections of ketamine rather than acepromazine did not produce this obesity; the weights of these rats were similar to those of normal littermates. These results indicate that a single injection of a neuroleptic during limbic seizures before puberty can produce neuronal alterations that contribute to a lifetime of obesity.

Radial maze proficiency of adult Wistar rats given prenatal complex magnetic field treatments.

Exposure to sinusoidal (power-frequency) magnetic fields during prenatal development is implicated in adulthood behavioral impairments. However, the effects of prenatal exposure to weak-intensity, nonsinusoidal complex magnetic fields (CMFs), an increasingly common feature of the modern environment, have not been rigorously examined. In the present study, male and female Wistar-strain rats were exposed continually during prenatal development to one of three extremely low-frequency CMFs or a sham condition. As adults, rats were trained in an acquisition/reversal radial maze task. All rats exposed to the prenatal CMFs increased their commission of reference memory errors, but differences in working memory and motivation to complete the maze task were specific to the type of prenatal CMF. These results provide the first evidence that prenatal exposures to specific shapes of CMFs impair complex learning behaviors into adulthood.

The brain matrix and multifocal brain damage following a single injection of ketamine in young adult rats: conspicuous changes in old age.

Male rats were seized with lithium and pilocarpine and then injected within 30 min. with either acepromazine or ketamine. These rats as well as age-matched normal rats were observed daily for one year. The rats which had received the ketamine after the seizures were significantly heavier than either the normal rats or the other group of seized rats. The bulk of this increased weight was due to the marked increase in white, extremely dense adipose tissue. Compared to the acepromazine-treated rats, the ketamine-treated rats did not exhibit spontaneous seizures and exhibited cerebral widths comparable to normal rats. These results suggest that the multifocal, graded neuronal loss associated with this seizure model may allow other "configurations" to emerge that can support normal behaviors as well as new characteristics.

Geophysical variables and behavior: XCI. Ambulatory behavior in rats following prenatal exposures to complex magnetic fields designed to interact with genetic expression.

A total of 45 litters were exposed during their entire prenatal development to one of two complex patterns of magnetic fields whose strengths varied within one of four intensity ranges between 10 nT and 1,000 nT or to sham-field conditions. The litters exposed to the most complex pattern, composed of 50 200-msec. presentations of different pulses for 10 sec. every 50 sec., displayed more ambulation in an open field at 21 days of age than the litters that had been exposed continuously to a repetitive frequency-modulated field or to sham-field conditions. This treatment explained 25% of the variance in the numbers of squares traversed. The results suggest that complexity of the applied magnetic field during prenatal development may be more important than intensity for permanently affecting neuronal organization and behavior.

Micellar electrokinetic chromatography separations of dynorphin peptide analogs.

Prodynorphin is a precursor that has multiple cleavage sites to release various dynorphin opioid peptides. The dynorphin analogs used in this study have 18 amino acid residues. A series of dynorphin-like peptides, differing by a single residue (alanine substitution) were assembled by Fmoc solid-phase procedures and purified by preparative high performance liquid chromatography (HPLC). Separation of the Ala-scan dynorphin analogs was investigated by micellar electrokinetic chromatography (MEKC) employing anionic, cationic and zwitterionic surfactants. The role of electrostatic and hydrophobic forces in analyte-surfactant interactions is discussed with respect to the observed elution patterns. Separation of all dynorphin analogs by MEKC using a zwitterionic surfactant shows this technique to be powerful for separating closely related peptide species. It also demonstrates the potential for using MEKC for the prescreening of peptide libraries to determine their biological activity toward specific receptors. Results from the separation of dynorphin analogs by free solution and ion-pairing capillary electrophoresis are also presented.

Inhibitors of V-type ATPases, bafilomycin A1 and concanamycin A, protect against beta-amyloid-mediated effects on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction.

The functional viability of cells can be evaluated using a number of different assay determinants. One common assay involves exposing cells to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which is converted intracellularly to a colored formazan precipitate and often used to assess amyloid peptide-induced cytotoxic effects. The MTT assay was employed to evaluate the role of endosomal uptake and lysosomal acidification in amyloid peptide-treated differentiated PC12 cell cultures using selective vacuolar-type (V-type) ATPase inhibitors. The macrolides bafilomycin A1 (BAF) and concanamycin A (CON) block lysosomal acidification through selective inhibition of the V-type ATPase. Treating nerve growth factor-differentiated PC12 cells with nanomolar concentrations of BAF or CON provides complete protection against the effects of beta-amyloid peptides Abeta(1-42), Abeta(1-40), and Abeta(25-35) and of amylin on MTT dye conversion. These macrolides do not inhibit peptide aggregation, act as antioxidants, or inhibit Abeta uptake by cells. Measurements of lysosomal acidification reveal that the concentrations of BAF and CON effective in reversing Abeta-mediated MTT dye conversion also reverse lysosomal pH. These results suggest that lysosomal acidification is necessary for Abeta effects on MTT dye conversion.

Geophysical variables and behavior: LXXXIV. Quantitative increases in group aggression in male epileptic rats during increases in geomagnetic activity.

Over a 24-day period the magnitude of the average, 24 hr., global geomagnetic activity was significantly correlated (r and rho about .40) with the average numbers of agonistic (biting) responses between midnight and 0100 hr. in a group of four males epileptic rats. Geomagnetic activity was not significantly correlated with quantitative measures of boxing, mounting, eating, grooming, or drinking. Partial correlations, which also controlled for a weak serial correlation, indicated that the index of geomagnetic activity was only associated with the biting (partial r = .54) behaviors. The results replicated previous studies and suggest that the relationship between aggression and geomagnetic activity is specific and not an artifact of a generalized increase in multiple domains of behavior.

Experimental induction of intermale aggressive behavior in limbic epileptic rats by weak, complex magnetic fields: implications for geomagnetic activity and the modern habitat?

In three separate experiments, groups (4/group) of male rats with limbic epilepsy were exposed for 80 min every 24 hr during the midscotophase for 24 successive days to sham-field conditions or to one of four complex patterns of magnetic fields whose average intensities ranged between 20 nT to 500 nT. The numbers of episodes of boxing, biting, mounting, eating, drinking and grooming were then recorded each night during the latter 20 min. Moderately strong statistically significant interactions occurred between the presence or absence of the field and the pattern of the field explained 25% and 50% of the variance in the numbers of biting and boxing responses, respectively. Other behaviors were not affected. The results suggest that group aggression can be increased or decreased as a function of the temporal characteristics and morphology (shape) of the applied magnetic field.