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Saxitoxin (STX) is a potent neurotoxin naturally produced by dinoflagellates and cyanobacteria. STX inhibits voltage-gated sodium channels (VGSCs), affecting the propagation of action potentials. Consumption of seafood contaminated with STX is responsible for paralytic shellfish poisoning (PSP). Humans are among the species most sensitive to PSP; neurological symptoms of exposure range from tingling of the extremities to severe paralysis. The objective of this study was to determine the effects of STX exposure on developmental processes during early embryogenesis. This study was designed to test the hypothesis that early developmental exposure to STX would disrupt key processes, particularly those related to neural development. Zebrafish embryos were exposed to STX (24 or 48pg) or vehicle (0.3mM HCl) at 6hours post fertilization (hpf) via microinjection. There was no overt toxicity but starting at 36 hpf there was a temporary lack of pigmentation in STX-injected embryos, which resolved by 72 hpf. Using high performance liquid chromatography, we found that STX was retained in embryos up to 72 hpf in a dose-dependent manner. Temporal transcriptional profiling of embryos exposed to 48pg STX per embryo revealed no differentially expressed genes (DEGs) at 24 hpf, but at 36 and 48 hpf, there were 3547 and 3356 DEGs, respectively. KEGG pathway analysis revealed significant enrichment of genes related to focal adhesion, adherens junction and regulation of actin cytoskeleton, suggesting that cell-cell and cell-extracellular matrix interactions were affected by STX. Genes affected are critical for axonal growth and the development of functional neural networks. We confirmed these findings by visualizing axonal defects in transgenic zebrafish with fluorescently labeled sensory neurons. In addition, our gene expression results suggest that STX exposure affects both canonical and noncanonical functions of VGSCs. Given the fundamental role of VGSCs in both physiology and development, these findings offer valuable insights into effects of exposure to neurotoxins., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Inertial measurement units (IMUs) are popular tools for estimating biomechanical variables such as peak vertical ground reaction force (GRF v ) and foot-ground contact time (t c ), often by using multiple sensors or predictive models. Despite their growing use, little is known about the effects of varying low-pass filter cutoff frequency, which can affect the magnitude of force-related dependent variables, the accuracy of IMU-derived metrics, or if simpler methods for such estimations exist. The purpose of this study was to investigate the effects of varying low-pass filter cutoff frequency on the correlation of IMU-derived peak GRF v and t c to gold-standard lab-based measurements. Thirty National Collegiate Athletics Association Division 1 cross country runners ran on an instrumented treadmill at a range of speeds while outfitted with a sacral-mounted IMU. A simple method for estimating peak GRF v from the IMU was implemented by multiplying the IMU's vertical acceleration by the runner's body mass. Data from the IMU were low-pass filtered with 5, 10, and 30 Hz cutoffs. Pearson correlation coefficients were used to determine how well the IMU-derived estimates matched gold-standard biomechanical estimations. Correlations ranged from very weak to moderate for peak GRF v and t c . For peak GRF v , the 10 Hz low-pass filter cutoff performed best (r = 0.638), while for t c the 5 Hz cut-off performed best (r = 0.656). These results suggest that IMU-derived estimates of force and contact time are influenced by the low-pass filter cutoff frequency. Further investigations are needed to determine the optimal low-pass filter cutoff frequency or a different method to accurately estimate force and contact time is suggested., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The forefoot functions as the base of support during late stance, rotating about the dual-axis of the metatarsophalangeal joints. Previous research has shown that joint axis definition affects estimated joint moments about the forefoot. However, little is known about how metatarsophalangeal joint center definition affects estimated joint kinetics. This study compared moments about the metatarsophalangeal joint using four different defined joint centers. There was a significant difference (p < .001) in peak moments between joint center definitions, differing by up to 0.488 N-m/kg for the slow and 0.878 N-m/kg for the fast running speeds tested. Additionally, there was a significant difference (p < .001) for when peak plantar flexor moment occurred during the slower running condition. The more posteriorly oriented joint centers resulted in higher moments and earlier onset of the plantar flexor moment. In addition to careful modeling of the metatarsophalangeal joint axis, it is recommended that joint center definition should be considered as well., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: End-stage ankle arthritis is a debilitating condition that often requires surgical intervention after failed conservative treatments. Ankle arthrodesis is a common surgical option, especially for younger and highly active patients; however, ankle arthroplasty has become increasingly popular as advancements in implant design improve device longevity. The longitudinal differences in biomechanical outcomes between these surgical treatments remain indistinct, likely due to the challenges associated with objective study of a heterogeneous population., Methods: Patients scheduled for arthroplasty (n = 27) and arthrodesis (n = 20) were recruited to participate in this three-year prospective study. Postoperative functional outcomes were compared at distinct annual time increments using measures of gait analysis, average daily step count and survey score., Findings: Both surgical groups presented reduced pain, improved survey scores, and increased walking speed at the first-year postoperative session, which were generally consistent across the three-year follow-up. Arthrodesis patients walked with decreased sagittal ankle RoM, increased sagittal hip RoM, increased step length, and increased transient force at heel strike, postoperatively. Arthroplasty patients increased ankle RoM and cadence, with no changes in hip RoM, step length or heel strike transient force., Interpretation: Most postoperative changes were detected at the first-year follow-up session and maintained across the three-year time period. Despite generally favorable outcomes associated with both surgeries, several underlying postoperative biomechanical differences were detected, which may have long-term functional consequences. Furthermore, neither technique was able to completely restore gait biomechanics to the levels of the contralateral unaffected limb, leaving potential for the development of improved surgical and rehabilitative treatments., (Published by Elsevier Ltd.)

6-Formylindolo[3,2-b]carbazole (FICZ) is a potent aryl hydrocarbon receptor (AHR) agonist that is efficiently metabolized by AHR-regulated cytochrome P4501 enzymes. FICZ is a proposed physiological AHR ligand that induces its own degradation as part of a regulatory negative feedback loop. In vitro studies in cells show that CYP1 inhibition in the presence of FICZ results in enhanced AHR activation, suggesting that FICZ accumulates in the cell when its metabolism is blocked. We used zebrafish (Danio rerio) embryos to investigate the in vivo effects of FICZ when CYP1A is knocked down or inhibited. Embryos were injected with morpholino antisense oligonucleotides targeting CYP1A (CYP1A-MO), Ahr2, or a combination of both. FICZ exposure of non-injected embryos or embryos injected with control morpholino had little effect. In CYP1A-MO-injected embryos, however, FICZ dramatically increased mortality, incidence and severity of pericardial edema and circulation failure, reduced hatching frequency, blocked swim bladder inflation, and strongly potentiated expression of Ahr2-regulated genes. These effects were substantially reduced in embryos with a combined knockdown of Ahr2 and CYP1A, indicating that the toxicity was mediated at least partly by Ahr2. Co-exposure to the CYP1 inhibitor alpha-naphthoflavone (αNF) and FICZ had similar effects as the combination of CYP1A-MO and FICZ. HPLC analysis of FICZ-exposed embryos showed increased levels of FICZ after concomitant CYP1A-MO injection or αNF co-exposure. Together, these results show that a functioning CYP1/AHR feedback loop is crucial for regulation of AHR signaling by a potential physiological ligand in vivo and further highlights the role of CYP1 enzymes in regulating biological effects of FICZ., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Increased practice difficulty during asymmetrical split-belt treadmill rehabilitation has been shown to improve gait outcomes during retention and transfer tests. However, research in this area has been limited by manual treadmill operation. In the case of variable practice, which requires stride-by-stride changes to treadmill belt velocities, the treadmill control must be automated. This paper presents a method for automation of asymmetrical split-belt treadmill walking, and evaluates how well this method performs with regards to timing of gait events. One participant walked asymmetrically for 100 strides, where the non-dominant limb was driven at their self-selected walking speed, while the other limb was driven randomly on a stride-by-stride basis. In the control loop, the key factors to insure that the treadmill belt had accelerated to its new velocity safely during the swing phase were the sampling rate of the A/D converter, processing time within the controller software, and acceleration of the treadmill belt. The combination of these three factors resulted in a total control loop time during each swing phase that satisfied these requirements with a factor of safety that was greater than 4. Further, a polynomial fit indicated that belt acceleration was the largest contributor to changes in this total time. This approach appears to be safe and reliable for stride-by-stride adjustment of treadmill belt speed, making it suitable for future asymmetrical split-belt walking studies. Further, it can be incorporated into virtual reality rehabilitation paradigms that utilize split-belt treadmill walking., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants. The most toxic PCBs are the non-ortho-substituted ("dioxin-like") congeners that act through the aryl hydrocarbon receptor (AHR) pathway. In humans, perinatal exposure to dioxin-like PCBs is associated with neurodevelopmental toxicity in children. Yet, the full potential for later-life neurobehavioral effects that result from early-life low level exposure to dioxin-like PCBs is not well understood. The objective of this study was to determine the effects of developmental exposure to low levels of dioxin-like PCBs on early- and later-life behavioral phenotypes using zebrafish as a model system. We exposed zebrafish embryos to either vehicle (DMSO) or low concentrations of PCB126 (0.3, 0.6, 1.2nM) for 20h (4-24h post fertilization), and then reared them to adulthood in clean water. Locomotor activity was tested at two larval stages (7 and 14 days post fertilization). Adult fish were tested for anxiety-related behavior using the novel tank and shoaling assays. Adult behavioral assays were repeated several times on the same group of fish and effects on intra- and inter-trial habituation were determined. While there was no effect of PCB126 on larval locomotor activity in response to changes in light conditions, developmental exposure to PCB126 resulted in impaired short- and long-term habituation to a novel environment in adult zebrafish. Cyp1a induction was measured as an indicator for AHR activation. Despite high induction at early stages, cyp1a expression was not induced in the brains of developmentally exposed adult fish that showed altered behavior, suggesting that AHR was not activated at this stage. Our results demonstrate the effectiveness of the zebrafish model in detecting subtle and delayed behavioral effects resulting from developmental exposure to an environmental contaminant., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Oxidative stress is an important mechanism of chemical toxicity, contributing to developmental toxicity and teratogenesis as well as to cardiovascular and neurodegenerative diseases and diabetic embryopathy. Developing animals are especially sensitive to effects of chemicals that disrupt the balance of processes generating reactive species and oxidative stress, and those anti-oxidant defenses that protect against oxidative stress. The expression and inducibility of anti-oxidant defenses through activation of NFE2-related factor 2 (Nrf2) and related proteins is an essential process affecting the susceptibility to oxidants, but the complex interactions of Nrf2 in determining embryonic response to oxidants and oxidative stress are only beginning to be understood. The zebrafish (Danio rerio) is an established model in developmental biology and now also in developmental toxicology and redox signaling. Here we review the regulation of genes involved in protection against oxidative stress in developing vertebrates, with a focus on Nrf2 and related cap'n'collar (CNC)-basic-leucine zipper (bZIP) transcription factors. Vertebrate animals including zebrafish share Nfe2, Nrf1, Nrf2, and Nrf3 as well as a core set of genes that respond to oxidative stress, contributing to the value of zebrafish as a model system with which to investigate the mechanisms involved in regulation of redox signaling and the response to oxidative stress during embryolarval development. Moreover, studies in zebrafish have revealed nrf and keap1 gene duplications that provide an opportunity to dissect multiple functions of vertebrate NRF genes, including multiple sensing mechanisms involved in chemical-specific effects., (Copyright © 2015. Published by Elsevier Inc.)

Results of recent studies showed that 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are equipotent in domestic chicken (Gallus gallus domesticus) while PeCDF is more potent than TCDD in ring-necked pheasant (Phasianus colchicus) and Japanese quail (Coturnix japonica). To elucidate the mechanism(s) underlying these differences in relative potency of PeCDF among avian species, we tested the hypothesis that this is due to species-specific differential binding affinity of PeCDF to the aryl hydrocarbon receptor 1 (AHR1). Here, we modified a cell-based binding assay that allowed us to measure the binding affinity of dioxin-like compounds (DLCs) to avian AHR1 expressed in COS-7 (fibroblast-like cells). The results of the binding assay show that PeCDF and TCDD bind with equal affinity to chicken AHR1, but PeCDF binds with greater affinity than TCDD to pheasant (3-fold) and Japanese quail (5-fold) AHR1. The current report introduces a COS-7 whole-cell binding assay and provides a mechanistic explanation for differential relative potencies of PeCDF among species of birds., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

Embryonic development involves dramatic changes in cell proliferation and differentiation that must be highly coordinated and tightly regulated. Cellular redox balance is critical for cell fate decisions, but it is susceptible to disruption by endogenous and exogenous sources of oxidative stress. The most abundant endogenous nonprotein antioxidant defense molecule is the tripeptide glutathione (γ-glutamylcysteinylglycine, GSH), but the ontogeny of GSH concentration and redox state during early life stages is poorly understood. Here, we describe the GSH redox dynamics during embryonic and early larval development (0-5 days postfertilization) in the zebrafish (Danio rerio), a model vertebrate embryo. We measured reduced and oxidized glutathione using HPLC and calculated the whole embryo total glutathione (GSHT) concentrations and redox potentials (Eh) over 0-120 h of zebrafish development (including mature oocytes, fertilization, midblastula transition, gastrulation, somitogenesis, pharyngula, prehatch embryos, and hatched eleutheroembryos). GSHT concentration doubled between 12h postfertilization (hpf) and hatching. The GSH Eh increased, becoming more oxidizing during the first 12h, and then oscillated around -190 mV through organogenesis, followed by a rapid change, associated with hatching, to a more negative (more reducing) Eh (-220 mV). After hatching, Eh stabilized and remained steady through 120 hpf. The dynamic changes in GSH redox status and concentration defined discrete windows of development: primary organogenesis, organ differentiation, and larval growth. We identified the set of zebrafish genes involved in the synthesis, utilization, and recycling of GSH, including several novel paralogs, and measured how expression of these genes changes during development. Ontogenic changes in the expression of GSH-related genes support the hypothesis that GSH redox state is tightly regulated early in development. This study provides a foundation for understanding the redox regulation of developmental signaling and investigating the effects of oxidative stress during embryogenesis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Non-articulated energy storage and return prosthetic feet lack any true articulation or obvious point of rotation. This makes it difficult to select a joint center about which to estimate their kinetics. Despite this absence of any clear point of rotation, methods for estimating the kinetic performance of this class of prosthetic feet typically assume that they possess a fixed center of rotation and that its location is well approximated by the position of the contralateral lateral malleolus. To evaluate the validity of this assumption we used a finite helical axis approach to determine the position of the center of rotation in the sagittal plane for a series of non-articulated energy storage and return prosthetic feet. We found that over the course of stance phase, the sagittal finite helical axis position diverged markedly from the typically assumed fixed axis location. These results suggest that researchers may need to review center of rotation assumptions when assessing prosthetic foot kinetics, while clinicians may need to reconsider the criteria by which they prescribe these prosthetic feet., (Published by Elsevier Ltd.)

Multixenobiotic transporters have been extensively studied for their ability to modulate the disposition and toxicity of pharmacological agents, yet their influence in regulating the levels of dietary toxins within marine consumers has only recently been explored. This study presents functional and molecular evidence for multixenobiotic transporter-mediated efflux activity and expression in the generalist gastropod Cyphoma gibbosum, and the specialist nudibranch Tritonia hamnerorum, obligate predators of chemically defended gorgonian corals. Immunochemical analysis revealed that proteins with homology to permeability glycoprotein (P-gp) were highly expressed in T. hamnerorum whole animal homogenates and localized to the apical tips of the gut epithelium, a location consistent with a role in protection against ingested prey toxins. In vivo dye assays with specific inhibitors of efflux transporters demonstrated the activity of P-gp and multidrug resistance-associated protein (MRP) families of ABC transporters in T. hamnerorum. In addition, we identified eight partial cDNA sequences encoding two ABCB and two ABCC proteins from each molluscan species. Digestive gland transcripts of C. gibbosum MRP-1, which have homology to vertebrate glutathione-conjugate transporters, were constitutively expressed regardless of gorgonian diet. This constitutive expression may reflect the ubiquitous presence of high affinity substrates for C. gibbosum glutathione transferases in gorgonian tissues likely necessitating export by MRPs. Our results suggest that differences in multixenobiotic transporter expression patterns and activity in molluscan predators may stem from the divergent foraging strategies of each consumer., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

The AHR is well known for regulating responses to an array of environmental chemicals. A growing body of evidence supports the hypothesis that the AHR also plays perhaps an even more important role in modulating critical aspects of cell function including cell growth, death, and migration. As these and other important AHR activities continue to be elucidated, it becomes apparent that attention now must be directed towards the mechanisms through which the AHR itself is regulated. Here, we review what is known of and what biological outcomes have been attributed to the AHR repressor (AHRR), an evolutionarily conserved bHLH-PAS protein that inhibits both xenobiotic-induced and constitutively active AHR transcriptional activity in multiple species. We discuss the structure and evolution of the AHRR and the dominant paradigm of a xenobiotic-inducible negative feedback loop comprised of AHR-mediated transcriptional up-regulation of AHRR and the subsequent AHRR-mediated suppression of AHR activity. We highlight the role of the AHRR in limiting AHR activity in the absence of xenobiotic AHR ligands and the important contribution of constitutively repressive AHRR to cancer biology. In this context, we also suggest a new hypothesis proposing that, under some circumstances, constitutively active AHR may repress AHRR transcription, resulting in unbridled AHR activity. We also review the predominant hypotheses on the molecular mechanisms through which AHRR inhibits AHR as well as novel mechanisms through which the AHRR may exert AHR-independent effects. Collectively, this discussion emphasizes the importance of this understudied bHLH-PAS protein in tissue development, normal cell biology, xenobiotic responsiveness, and AHR-regulated malignancy.

Many studies have investigated the relationships between electromyography (EMG) and torque production. A few investigators have used adjusted learning algorithms and feed-forward artificial neural networks (ANNs) to estimate joint torque in the elbow. This study sought to estimate net isokinetic knee torque using ANN models. Isokinetic knee extensor and flexor torque data were measured simultaneously with agonist and antagonist EMG during concentric and eccentric contractions at joint velocities of 30 degrees /s and 60 degrees /s. Age, gender, height, body mass, agonist EMG, antagonist EMG, joint position and joint velocity were entered as predictive variables of net torque. A three-layer ANN model was developed and trained using an adjusted back-propagation algorithm. Accuracy results were compared against those of forward stepwise regression models. Stepwise regression models included body mass, body height and joint position as the most influential predictors, followed by agonist EMG for concentric and eccentric contractions. Estimation of eccentric torque included antagonist EMG following the agonist activation. ANN models resulted in more accurate torque estimation (R=0.96), compared to the stepwise regression models (R=0.71). ANN model accuracy increased greatly when the number of hidden units increased from 5 to 10, continuing to increase gradually with additional hidden units. The average number of training epochs necessary for solution convergence and the relative accuracy of the model indicate a strong ability for the ANN model to generalize these estimations to a broader sample. The ANN model appears to be a feasible technique for estimating joint torque in the knee.

The aryl hydrocarbon receptor (AHR) and AHR repressor (AHRR) proteins regulate gene expression in response to some halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons. The Atlantic killifish is a valuable model of the AHR signaling pathway, but antibodies are not available to fully characterize AHR and AHRR proteins. Using bacterially expressed AHRs, we developed specific and sensitive polyclonal antisera against the killifish AHR1, AHR2, and AHRR. In immunoblots, these antibodies recognized full-length killifish AHR and AHRR proteins synthesized in rabbit reticulocyte lysate, proteins expressed in mammalian cells transfected with killifish AHR and AHRR constructs, and AHR proteins in cytosol preparations from killifish tissues. Killifish AHR1 and AHR2 proteins were detected in brain, gill, kidney, heart, liver, and spleen. Antisera specifically precipitated their respective target proteins in immunoprecipitation experiments with in vitro-expressed proteins. Killifish ARNT2 co-precipitated with AHR1 and AHR2. These sensitive, specific, and versatile antibodies will be valuable to researchers investigating AHR signaling and other physiological processes involving AHR and AHRR proteins.

Gait patterns of the elderly are often adjusted to accommodate for reduced function in the balance control system and a general reduction in skeletal muscle strength. Recent studies have demonstrated that measures related to motion of whole body center of mass (COM) can distinguish elderly individuals with balance impairment from healthy peers. Accurate COM estimation requires a multiple-segment anthropometric model, which may restrict its broad application in assessment of dynamic instability. Although temporal-distance measures and electromyography have been used in evaluation of overall gait function and determination of gait dysfunction, no studies have examined the use of gait measurements in predicting COM motion during gait. The purpose of this study was to demonstrate the effectiveness of an artificial neural network (ANN) model in mapping gait measurements onto COM motion in the frontal plane. Data from 40 subjects of varied age and balance impairment were entered into a 3-layer feed-forward model with back-propagated error correction. Bootstrap re-sampling was used to enhance the generalization accuracy of the model, using 20 re-sampling trials. The ANN model required minimal processing time (5 epochs, with 20 hidden units) and accurately mapped COM motion (R-values up to 0.89). As training proportion and number of hidden units increased, so did model accuracy. Overall, this model appears to be effective as a mapping tool for estimating balance control during locomotion. With easily obtained gait measures as input and a simple, computationally efficient architecture, the model may prove useful in clinical scenarios where electromyography equipment exists.

Accidental falls are a leading cause of injury and death in the growing elderly population. Traumatic falls are frequent, costly, and debilitating. Control of balance during locomotion is critical for safe ambulation, but relatively little is known about the natural effect of aging on dynamic balance control. Samples of healthy young (n = 13) and elderly (n = 13) subjects were compared in the interactive measures of center of mass (COM) and center of pressure (COP) during level walking and obstacle crossing conditions. Obstacle heights were normalized to individual body height (2.5%, 5%, 10%, and 15%). Temporal-distance (T-D) variables of gait were also compared. Statistical analyses were conducted using a two-way ANOVA for subject group and obstacle height. T-D parameters were not significantly different between groups; nor were frontal plane COM and COP parameters. Significant age differences did exist for antero-posterior (A/P) motion of the COM (decreased motion in the elderly), and its relationship with the COP (reduced separation between the two variables in the elderly). Anterior COM velocities were also significantly lower in the elderly group. The results confirm the ability of healthy elderly adults to maintain dynamic balance control in the frontal plane during locomotion. Reduced A/P distances between the COM and COP indicate a conservative reduction of the mechanical load on joints of the supporting limb. This conservative strategy may be related to a reduction in muscle strength as it occurs in the natural aging process.

Objective: To determine if medio-lateral motion of the head, trunk, or pelvis demonstrates dynamic stability as well as whole-body center of mass during obstructed walking., Design: Group comparison of two elderly populations using whole-body motion analysis., Background: Detection of imbalance through analysis of center of mass motion is commonly adopted, requiring three-dimensional reconstruction of a multi-link biomechanical model. It would be advantageous clinically if similar detection could be made by analyzing segmental displacements of the pelvis, trunk, or head., Methods: Healthy elderly adults and elderly patients with balance disorders walked over level ground and crossed obstacles of height ranging from 2.5% to 15% of body height. Whole-body center of mass was calculated as the weighted sum of segmental centers of mass. Group differences in medio-lateral displacements and peak velocities of head, trunk, pelvis, and the center of mass were analyzed using a two-way ANOVA with repeated measures for obstacle height., Results: Elderly patients with balance disorders exhibited greater medio-lateral displacement and peak velocities of all segments. However, significant group differences were only detected in the center of mass displacement and peak velocity., Conclusion: Whole-body center of mass motion distinguishes elderly patients with balance disorders from healthy peers more consistently than markers representing the head, trunk, or pelvis. Large variation of individual segment motion makes dynamic stability difficult to assess. This study demonstrates that center of mass motion allows more sensitive detection of dynamic instability., Relevance: Detection of dynamic instability in at-risk individuals before falls occur will allow preventative interventions, preserving quality of life in the elderly population.

Changes in the expression of the aryl hydrocarbon receptor (AHR) have been documented in several systems and in response to a variety of treatments. The significance of these findings is unclear, because the effects of such changes on subsequent responses to AHR ligands seldom have been measured. We tested the ability of changes in serum used in cell culture medium to alter expression of the AHR and induction of cytochrome P4501A (CYP1A) in PLHC-1 teleost hepatoma cells. Culture of early-passage cells in serum-free medium for 2 days led to a loss of CYP1A inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast, culture in 10% delipidated calf serum increased the TCDD-induced levels of both CYP1A protein and enzymatic activity relative to levels in cells cultured in 10% complete calf serum. These effects were consistent between 8 and 24hr post-treatment, indicating that the kinetics of induction were unaffected. In cells cultured in serum-free medium for 1 and 2 days there was a progressive loss of CYP1A inducibility. This loss of response paralleled a time-dependent decline in AHR protein, as measured by specific binding of [3H]TCDD. Using an operational model for AHR action in PLHC-1 cells, the measured reduction in AHR could be shown to predict the loss of CYP1A induction. Expression of AHR protein was unaffected by culture in 10% delipidated serum. The effects of serum-free medium and delipidated serum were found only in early-passage cells; inducibility of CYP1A and expression of AHR protein in late-passage cells were unaffected by serum withdrawal. Comparison of early- and late-passage cells revealed a 2-fold greater rate of proliferation in the latter, suggesting that a growth advantage is coincident with loss of the serum-dependency of AHR expression. These results provide a quantitative link between changes in receptor expression and a downstream response, establishing a foundation for future studies of receptor expression and sensitivity to toxic responses in vitro and in vivo.

We measured rates of oxidative metabolism of two tetrachlorobiphenyl (TCB) congeners by hepatic microsomes of two marine mammal species, beluga whale and pilot whale, as related to content of selected cytochrome P450 (CYP) forms. Beluga liver microsomes oxidized 3,3',4,4'-TCB at rates averaging 21 and 5 pmol/min per mg for males and females, respectively, while pilot whale samples oxidized this congener at 0.3 pmol/min per mg or less. However, rates of 3,3',4,4'-TCB metabolism correlated with immunodetected CYP1A1 protein content in liver microsomes of both species. The CYP1A inhibitor alpha-naphthoflavone inhibited 3,3',4,4'-TCB metabolism by 40% in beluga, supporting a role for a cetacean CYP1A as a catalyst of this activity. Major metabolites of 3,3',4,4'-TCB generated by beluga liver microsomes were 4-OH-3,3',4',5-TCB and 5-OH-3,3',4,4'-TCB (98% of total), similar to metabolites formed by other species CYP1A1, and suggesting a 4,5-epoxide-TCB intermediate. Liver microsomes of both species metabolized 2,2',5,5'-TCB at rates of 0.2-1.5 pmol/min per mg. Both species also expressed microsomal proteins cross-reactive with antibodies raised against some mammalian CYP2Bs (rabbit; dog), but not others (rat; scup). Whether CYP2B homologues occur and function in cetaceans is uncertain. This study demonstrates that PCBs are metabolized to aqueous-soluble products by cetacean liver enzymes, and that in beluga, rates of metabolism of 3,3',4,4'-TCB are substantially greater than those of 2,2',5,5'-TCB. These directly measured rates generally support the view that PCB metabolism plays a role in shaping the distribution patterns of PCB residues found in cetacean tissue.

The aryl hydrocarbon receptor (Ah receptor or AHR) is a ligand-activated transcription factor involved in the regulation of several genes, including those for xenobiotic-metabolizing enzymes such as cytochrome P450 1A and 1B forms. Ligands for the AHR include a variety of aromatic hydrocarbons, including the chlorinated dioxins and related halogenated aromatic hydrocarbons whose toxicity occurs through activation of the AHR. The AHR and its dimerization partner ARNT are members of the emerging bHLH-PAS family of transcriptional regulatory proteins. In this review, our current understanding of the AHR signal transduction pathway in non-mammalian and other non-traditional species is summarized, with an emphasis on similarities and differences in comparison to the AHR pathway in rodents and humans. Evidence and prospects for the presence of a functional AHR in early vertebrates and invertebrates are also examined. An overview of the bHLH-PAS family is presented in relation to the diversity of bHLH-PAS proteins and the functional and evolutionary relationships of the AHR and ARNT to the other members of this family. Finally, some of the most promising directions for future research on the comparative biochemistry and molecular biology of the AHR and ARNT are discussed.

The mammalian aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that controls the expression of cytochrome P450 1A (CYP1A) genes in response to halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The natural ligand and normal physiologic function of this protein are as yet unknown. One approach to understanding AHR function and significance is to determine the evolutionary history of this receptor and of processes such as CYP1A induction that are controlled by the AHR in mammals. In these studies, AHR function was evaluated in representative cartilaginous fish (little skate, Raja erinacea) and jawless fish (sea lamprey, Petromyzon marinus and Atlantic hagfish, Myxine glutinosa), using CYP1A induction as a model AHR-dependent response. Treatment of skate with beta-naphthoflavone (BNF) caused an 8-fold increase in hepatic ethoxyresorufin O-deethylase (EROD) activity as well as a 37-fold increase in the content of immunodetectable CYP1A protein. Evidence of CYP1A inducibility was also obtained for another cartilaginous fish, the smooth dogfish Mustelus canis. In contrast, hepatic EROD activity was not detected in untreated lamprey nor in lamprey treated with 3,3'4,4'-tetrachlorobiphenyl (TCB), a potent AHR agonist in teleosts. A possible CYP1A homolog was detected in lamprey hepatic microsomes by one of three antibodies to teleost CYP1A, but expression of this protein was not altered by TCB treatment. CYP1A protein and catalytic activity were measurable in hagfish, but neither was induced after treatment with TCB. These results suggest that the AHR-CYP1A signal transduction pathway is highly conserved in gnathostomes, but that there may be fundamental differences in AHR signaling or AHR-CYP1A coupling in agnathan fish. Agnathan fish such as hagfish and lamprey may be interesting model species for examining possible ancestral AHR functions not related to CYP1A regulation.

Concentration-dependent induction of cytochrome P4501A (CYP1A) and intracellular porphyrin accumulation were observed following treatment of chicken embryo hepatocyte (CEH) cultures with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 3,3',4,4'-tetrachlorobiphenyl (PCB 77, IUPAC nomenclature), 2,3',4,4',5-pentachlorobiphenyl (PCB 118), 3,3',4,4',5-pentachlorobiphenyl (PCB 126), 3,3',4,4',5,5'-hexachlorobiphenyl (PCB 169), and a commercial mixture of PCBs (Aroclor 1254). For these halogenated aromatic hydrocarbons (HAHs), or mixture, maximal CYP1A activity [measured as ethoxyresorufin-O-deethylase (EROD) activity] and immunodetectable protein were observed at concentrations just prior to, or coincident with, the concentrations at which porphyrin accumulation became evident. Both immunodetectable CYP1A protein and catalytic activity decreased at high concentrations of these compounds, but the rate and extent of decrease of immunodetectable CYP1A protein varied. Time-course studies with PCB 77 indicated a decrease in potency and an increase in maximal CYP1A induction between 24 and 48 hr of exposure which may indicate in vitro metabolism of this HAH. Intracellular accumulation of total porphyrins without CYP1A induction, was observed for 2,2',5,5'-tetrachlorobiphenyl (PCB 52), 2,2',6,6'-tetrachlorobiphenyl (PCB 54), 2,2',3,5',6-pentachlorobiphenyl (PCB 95), 2,2',4,5,5'-pentachlorobiphenyl (PCB 101), 2,2',3,3',6,6'-hexachlorobiphenyl (PCB 136), and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153). Overall, these results are consistent with a role for CYP1A induction and/or Ah receptor activation in porphyrin accumulation mediated by HAHs with a planar configuration, whereas those that are not planar may mediate porphyrin accumulation by a mechanism not involving induction of CYP1A.

The ubiquity of lead in our environment, its toxic nature and its potential to alter behavior of humans and animals has stimulated much research. In addition to the well known, but complex, changes in activity and performance of learned tasks following exposure to lead, an increasing body of literature suggests that changes in social behavior also occur. This study examined the impact of ingesting a 0.5% lead acetate solution (as the only available fluid)--a protocol that results in ca. 100 micrograms/dl blood-levels in our Binghamton Heterogenous (HET) mice--on food competition in both male and female HET mice. Effects of this lead exposure on cricket predation by the same HET mice also were observed. Our results from the food competition study (Experiment I) show, compared with water controls, that such exposure to lead increases both food possession time and amount of social contact after food consumption; but very little agonistic behavior took place in either the water control or lead-exposed competition testing sessions. In the cricket predation observations (Experiment II) there was a tendency for lead exposure to reduce the latency of males to attack, and the indication that lead-exposed females initially attacked a cricket's legs more often than any of the other groups. In fact, suggestions of such gender x treatment interactions occurred in both the food competition and the predation work. Overall, we believe our results, in conjunction with other relevant literature, suggest that exposure to lead reduced the individual's general fitness, even when levels are relatively low.