HESI workshop summary: Interpretation of developmental and reproductive toxicity endpoints and the impact on data interpretation of adverse events.

The Health and Environmental Sciences Institute Developmental and Reproductive Toxicology (HESI-DART) group held a hybrid in-person and virtual workshop in Washington, DC, in 2022. The workshop was entitled, "Interpretation of DART in Regulatory Contexts and Frameworks." There were 154 participants (37 in person and 117 virtual) across 9 countries. The purpose of the workshop was to capture key consensus approaches used to assess DART risks associated with chemical product exposure when a nonclinical finding is identified. The decision-making process for determining whether a DART endpoint is considered adverse is critical because the outcome may have downstream implications (e.g., increased animal usage, modifications to reproductive classification and pregnancy labeling, impact on enrollment in clinical trials and value chains). The workshop included a series of webinar modules to train and engage in discussions with federal and international regulators, clinicians, academic investigators, nongovernmental organizations, contract research organization scientists, and private sector scientists on the best practices and principles of interpreting DART and new approach methodologies in the context of regulatory requirements and processes. Despite the differences in regulatory frameworks between the chemical and pharmaceutical sectors, the same foundational principles for data interpretation should be applied. The discussions led to the categorization of principles, which offer guidance for the systematic interpretation of data. Step 1 entails identifying any hazard by closely analyzing the data at the study endpoint level, while Step 2 involves assessing risk using weight of evidence. These guiding principles were derived from the collective outcomes of the workshop deliberations.

Development of micro-electrode array based tests for neurotoxicity: assessment of interlaboratory reproducibility with neuroactive chemicals.

Neuronal assemblies within the nervous system produce electrical activity that can be recorded in terms of action potential patterns. Such patterns provide a sensitive endpoint to detect effects of a variety of chemical and physical perturbations. They are a function of synaptic changes and do not necessarily involve structural alterations. In vitro neuronal networks (NNs) grown on micro-electrode arrays (MEAs) respond to neuroactive substances as well as the in vivo brain. As such, they constitute a valuable tool for investigating changes in the electrophysiological activity of the neurons in response to chemical exposures. However, the reproducibility of NN responses to chemical exposure has not been systematically documented. To this purpose six independent laboratories (in Europe and in USA) evaluated the response to the same pharmacological compounds (Fluoxetine, Muscimol, and Verapamil) in primary neuronal cultures. Common standardization principles and acceptance criteria for the quality of the cultures have been established to compare the obtained results. These studies involved more than 100 experiments before the final conclusions have been drawn that MEA technology has a potential for standard in vitro neurotoxicity/neuropharmacology evaluation. The obtained results show good intra- and inter-laboratory reproducibility of the responses. The consistent inhibitory effects of the compounds were observed in all the laboratories with the 50% Inhibiting Concentrations (IC(50)s) ranging from: (mean ± SEM, in µM) 1.53 ± 0.17 to 5.4 ± 0.7 (n = 35) for Fluoxetine, 0.16 ± 0.03 to 0.38 ± 0.16 µM (n = 35) for Muscimol, and 2.68 ± 0.32 to 5.23 ± 1.7 (n = 32) for Verapamil. The outcome of this study indicates that the MEA approach is a robust tool leading to reproducible results. The future direction will be to extend the set of testing compounds and to propose the MEA approach as a standard screen for identification and prioritization of chemicals with neurotoxicity potential.

Complete inhibition of spontaneous activity in neuronal networks in vitro by deltamethrin and permethrin.

Types I and II pyrethroid insecticides cause temporally distinct decreases in voltage-gated sodium channel (VGSC) inactivation rates that are proposed to underlie their characteristic differences in toxicity signs. How alterations in VGSC channel function give rise to the characteristic differences in signs of pyrethroid intoxication is not completely understood, particularly those changes that occur in functional networks of interconnected neurons. To characterize better pyrethroid actions at the network level, effects of the Type I pyrethroid permethrin (PM) and the Type II pyrethroid deltamethrin (DM) on spontaneous glutamate network-dependent spikes and bursts were investigated in primary cultures of frontal cortex or spinal cord neurons grown on microelectrode arrays (MEAs). Fast GABAergic transmission was blocked by BIC, and concentration-dependent effects of DM (1nM to 5microM) and PM (10nM to 50microM) were examined. Both compounds caused concentration-dependent reductions in the network spike and burst rates. DM was more potent than PM, with IC(50) values of approximately 0.13 and approximately 4microM for inhibition of spike rate in cortical and spinal cord neurons, respectively. Both compounds decreased the percentage of spikes that occurred within a burst and increased the interspike interval within bursts. Onset of effects was rapid, but recovery from total activity loss was not readily achievable. Individual neurons responded heterogeneously; activity of most declined monophasically, but activity in others exhibited biphasic responses with increases followed by decreases in activity. In spinal cord, DM caused a greater number of biphasic responses (29%) than PM (10%). These results demonstrate that both DM and PM inhibit activity of glutamatergic networks, but with different potencies.

Accumulation of methylmercury or polychlorinated biphenyls in in vitro models of rat neuronal tissue.

In vivo exposure levels for neurotoxicants are often reported in parts per million (ppm) concentration in tissue, whereas exposure levels in experiments utilizing in vitro models are most commonly reported in micromolar (muM) concentration in the exposure solution. The present experiments sought to determine whether or not in vitro solution concentration was an appropriate dose-metric for comparison to in vivo tissue levels for lipophilic compounds. To do so, the accumulation of the polychlorinated biphenyl (PCB) mixture Aroclor 1254 (A1254) or methylmercury (MeHg) was examined in three commonly utilized in vitro neuronal tissue models: nerve growth factor differentiated pheochromocytoma (PC12) cells, primary cultures of rat neocortical cells, and adult rat hippocampal slices. Tissues were exposed to A1254 (0.65 ppm) or to MeHg (0.0033-0.33 ppm) in serum-free media for 1 or 24 h. Total PCB or mercury accumulation was measured by dual column gas chromatography with electron capture detection or by cold vapor atomic absorption, respectively. PC12 cells accumulated 66.7 and 103.8 ppm PCBs after 1 and 24 h exposure to A1254. Neocortical neurons also accumulated significant concentrations of PCBs, but less so than PC12 cells. After 1 h exposure to 0.65 ppm A1254, slices contained 3.46 and 0.81 ppm PCBs when exposed in a static and perfused system, respectively. After 1 h exposure to 0.0033, 0.033, and 0.33 ppm MeHg, PC12 cells contained 0.3, 2.2, and 17.7 ppm mercury, respectively; after 24 h, PC12 cells contained 0.4, 2.8, and 21.9 ppm. Hippocampal slices accumulated 1.7 and 4.8 ppm mercury after 1 and 3 h exposure to 0.33 ppm MeHg. For comparison, mercury accumulation in rat fetal and pup brain tissue after maternal exposure [0, 0.1, 1.0, or 2.0 mg/kg/day MeHg from gestational day (GD) 6-15] ranged from 0.05 to 7.89 ppm in 0.1 mg/kg dose animals on postnatal day 10 and 2.0 mg/kg dose animals on GD16, respectively. These results demonstrate that accumulation of PCBs and MeHg in vitro is tissue-, time-, and concentration-dependent and indicates that tissue levels rather than exposure concentrations are a more appropriate metric for comparison of in vitro to in vivo effects.

Identification of calcium-dependent and -independent signaling pathways involved in polychlorinated biphenyl-induced cyclic AMP-responsive element-binding protein phosphorylation in developing cortical neurons.

Cyclic AMP (cAMP)-responsive element-binding protein (CREB) is a transcription factor important in developing nervous system cells and is activated by a variety of signaling molecules. Aroclor 1254 (A1254), a polychlorinated biphenyl mixture, perturbs Ca(2+) homeostasis and increases CREB phosphorylation in rat neonatal cortical cell cultures in a time- and concentration-dependent manner. The present experiments determined that the cell type responding to A1254 with Ca(2+) increases and phosphorylated CREB (phospho-CREB) was predominantly of neuronal morphology and microtubule-associated protein (MAP2)-positive phenotype. Similarly, glutamate (100 microM) increased phospho-CREB immunoreactivity selectively in MAP2-immunopositive cells. Using Western blotting and immunocytochemical techniques, we identified key signal transduction pathways operative in phosphorylating CREB in cortical cell cultures and examined their participation in 3 ppm A1254-induced CREB activation. Cortical cultures treated with glutamate, forskolin or the phorbol ester phorbol 12-myristate 13-acetate exhibited robust increases in phospho-CREB. Tetrodotoxin (1 microM) completely inhibited CREB phosphorylation by A1254, suggesting that synaptic activity is involved in A1254-induced CREB activation. Buffering [Ca(2+)](i) with bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester in the absence of extracellular Ca(2+) partially inhibited A1254-induced CREB phosphorylation. Inhibition of mitogen-activated protein kinase (10 microM U0126) or protein kinase C (PKC; bisindoylmaleimide, 5 microM) activation did not inhibit A1254-induced CREB phosphorylation. By contrast, inhibition of protein kinase A (PKA) with 100 microM PKA inhibitor peptide, PKI, blocked A1254-induced CREB phosphorylation. Thus, we examined whether A1254 activates PKA by increasing cAMP; 10 microM forskolin, but not A1254, elevated intracellular cAMP levels. These results indicate that in neocortical cells in culture, CREB phosphorylation occurs via Ca(2+)-, PKA-, and PKC-dependent pathways. Furthermore, A1254-induced CREB phosphorylation occurs predominantly in neurons, is dependent on synaptic activity and mediated by Ca(2+)- and PKA-dependent pathways.

Inositol 1,4,5-triphosphate receptor-sensitive Ca(2+) release, store-operated Ca(2+) entry, and cAMP responsive element binding protein phosphorylation in developing cortical cells following exposure to polychlorinated biphenyls.

The present study assessed intracellular Ca(2+) signaling pathways sensitive to polychlorinated biphenyls (PCBs), xenobiotics that perturb neural development and plasticity. Mobilization of intracellular Ca(2+) stores after acute exposure to a PCB mixture, Aroclor 1254 (A1254), as well as selected PCB congeners, was studied in P0 rat cortical neuronal culture using fluorescence microscopy. Ca(2+) responses to A1254 progressed from a transient intracellular Ca(2+) increase (lasting 3--5 min) at 1 to 2 microM (0.3-0.6 ppm) to a Ca(2+) transient with store-operated Ca(2+) influx and later disturbances of basal Ca(2+) concentration; this latter pattern occurred more often with 10 to 20 microM (3--6 ppm) A1254. Thapsigargin, xestospongin C, and carbachol/Ca(2+)-free buffer blocked significantly the PCB-induced Ca(2+) transient, whereas both ryanodine (to deplete ryanodine-sensitive stores) and the L-type Ca(2+) channel blocker nifedipine were without effect on the A1254 initial Ca(2+) transient. Both thapsigargin and xestospongin also blocked latent elevations (at 0.5 h) in Ca(2+), disturbances that depend upon extracellular Ca(2+) entry via ion channels. Two possible consequences were explored. Phosphorylation of cAMP responsive element binding protein, a Ca(2+)-activated nuclear transcription factor (CREB), occurred in an A1254 concentration-dependent manner and persisted at least 1 h. Cell viability following a 24-h exposure to A1254 (2-20 microM) was decreased at 20 microM, but only in cells cultured >6 days. This cell death did not occur via an apoptotic mechanism. These results indicate that Ca(2+) disturbances following PCB exposure are associated with 1) discrete alterations in IP(3) receptor-mediated signals and 2) activation of downstream events that impact developing cortical cells.

Neurotoxicological outcomes of perinatal heptachlor exposure in the rat.

The developing nervous system has been identified as a potential target of pesticide exposure. Heptachlor is a cyclodiene pesticide that was widely used for many years, and for which inadvertent exposure to children and fetuses took place in the early 1980s; yet little is known regarding the developmental neurotoxicity of it and other cyclodienes. The aim of this study was to determine whether perinatal heptachlor exposure results in persistent alterations in nervous system function. Pregnant Sprague-Dawley dams were dosed from gestational day (GD) 12 to postnatal day (PND) 7, whereupon the rat pups were dosed directly until PND 21 (group A) or PND 42 (group B). Dose levels were 0, 0.03, 0.3, or 3 mg/kg/day, po. There were no dose-related effects on maternal weight, litter size, or pup growth. GABA(A) receptor binding (using [(35)S] tert-butylbicyclophosphorothionate; TBPS) and GABA-stimulated Cl- flux were evaluated in control and high-dose brain tissues taken on PND 7, 21, and 43. The B(max) values for [(35)S]-TBPS binding in brainstem, but not cortex, were decreased in female rats across all ages tested. There were no such changes in male rats, nor were K(D) values altered in either tissue or gender. GABA-stimulated Cl- flux was decreased in female cortex synaptoneurosomes only on PND 21. The ontogeny of the righting response (PND 2-5) was delayed in the high-dose females. All subsequent testing took place a week to months after dosing ceased. The functional observational battery (FOB) showed treatment-related, but not necessarily dose-related, changes in different aspects of the rat's reactivity and activity levels. Group-A rats also showed altered within-session habituation of motor activity. There were no heptachlor-related differences in motor activity following challenge with a range of chlordiazepoxide doses. Cognitive assessments were conducted in both groups of rats. There were no statistically significant differences among treatment groups in a one-trial passive avoidance test, although there was a trend toward less learning. In group B, rats (both sexes), heptachlor altered spatial learning in the Morris water maze during two weeks of daily training (2 trials/day). On probe trials, heptachlor-treated rats did not show significant preference for the correct quadrant (all dose groups in males, high dose in females). These rats did not show alterations on subsequent working-memory training (where the platform position was relearned each day). Thus, perinatal exposure to heptachlor produced neurochemical and persistent neurobehavioral changes, including alterations in spatial learning and memory.

Polychlorinated biphenyl-stimulation of Ca(2+) oscillations in developing neocortical cells: a role for excitatory transmitters and L-type voltage-sensitive Ca(2+) channels.

Developmental exposure to polychlorinated biphenyls (PCBs), environmental toxicants found throughout the world, results in neurodevelopmental delays and/or deficits. Previous mechanistic studies have demonstrated that PCBs elicit a broad spectrum of biochemical responses that include slow, graded increases in intracellular Ca(2+). Acute exposure of cultures of newborn rodent cortical neurons to the commercial PCB mixture Aroclor 1254 [A1254; 1-20 microM (0.3-6 ppm)], induced recurring oscillations of intracellular Ca(2+) concentration (individual Ca(2+) amplitudes of 200-600 nM). This oscillatory activity was absent in control (0.5 mM Mg(2+)-containing) solution. Ca(2+) oscillations induced by a 1-h exposure to A1254 were concentration dependent, as measured by cell recruitment (proportion of responding cells) as well as by Ca(2+) oscillation frequency and amplitude. Extracellular Ca(2+) entry via L-type voltage-sensitive Ca(2+) channels (VSCCs) was required to elicit the Ca(2+) oscillations because oscillations induced by A1254 were blocked in Ca(2+)-deficient solution or by addition of 1 microM nifedipine. Tetrodotoxin also blocked the Ca(2+) oscillations, suggesting that synaptic activity may activate VSCCs. To examine this further, the role of postsynaptic receptors that indirectly activate L-type VSCCs was examined. At 4 to 5 days in vitro, when GABA exerts a depolarizing action and activates L-type channels, addition of bicuculline blocked Ca(2+) oscillations induced by A1254. After longer maintenance of the cells in vitro (7 days), A1254-induced Ca(2+) oscillations were selectively blocked by a combination of N-methyl-D-aspartate and non-N-methyl-D-aspartate receptor antagonists (D-2-amino-5-phosphonopentanoic acid and 2, 3-dihydroxy-6,7-dinitroquinoxaline, respectively). These novel findings show the induction of network activity in an in vitro model by A1254 via activation of excitatory GABAergic and/or glutamatergic synaptic activity, depending on the stage of maturation.

Methylmercury effects on ion channels and electrical activity in neurons: future directions.

Methylmercury (CH3Hg+) is a potent neurotoxicant in humans and laboratory animals, and both epidemiological and laboratory data suggest that the developing nervous system is more susceptible to CH3Hg+ neurotoxicity than is the adult nervous system. While it is recognized that the developing nervous system is more susceptible to CH3Hg+ neurotoxicity compared to the adult nervous system, it is presently not clear what level of exposure, if any, is without effect on the developing human nervous system. A better understanding of mechanisms of action of CH3Hg+ for developmental neurotoxicity would be useful in defining risks associated with CH3Hg+ exposure. While alterations in a variety of processes may contribute to the neurotoxicity of CH3Hg+, changes in ion channel function and electrical activity in neuronal cells is a consistent observation following acute exposure in a variety of preparations. Additional research, however, is needed to clarify the relationship between alterations in neuronal electrophysiological function and developmental neurotoxicity of CH3Hg+. This article suggests several issues to be considered in order to address the relationship between in vitro acute effects of CH3Hg+ on ion channels and electrophysiological function in neurons and developmental neurotoxicity. Future studies need: 1) to examine effects on ion channel function and neuronal electrophysiology following subacute and chronic in vitro exposure to CH3Hg+; 2) to utilize model systems which consider developmental changes in neuronal function; 3) to consider direct vs. indirect effects of CH3Hg+; 4) to compare in vitro to ex vivo and in vivo effects; 5) to utilize in vitro dose levels which reflect in vivo exposure, and 6) to consider interactions between CH3Hg+ and other potential neurotoxicants found in environmental mixtures. Ultimately, it may be possible to develop biologically-based dose-response models of CH3Hg+ neurotoxicity which will be useful in assessing the risks of developmental neurotoxicity of this metal.

Perturbation by the PCB mixture aroclor 1254 of GABA(A) receptor-mediated calcium and chloride responses during maturation in vitro of rat neocortical cells.

GABA(A) receptors are targets of highly chlorinated environmental chemicals and have important roles in developing neurons. As such, we examined effects of polychlorinated biphenyls (PCBs) on GABA(A) receptor responses in primary cultures of rat neocortical cells using fluorescence imaging techniques. Between days in vitro (DIV) 5 and 8, the effect of GABA(A) receptor stimulation switched from excitatory (Ca(2+) entry following a Cl(-) efflux; DIV /=7). GABA(A)-receptor-stimulated increases in [Ca(2+)](i) were diminished in a concentration-dependent (1-20 microM) manner following 1 h of exposure to the PCB mixture Aroclor 1254 (A1254), with significant reductions at concentrations as low as 2 microM. A1254 (1-20 microM) also led to concentration-dependent increases in basal [Ca(2+)](i), irrespective of DIV. A1254 (10 and 20 microM) significantly increased basal Ca(2+)(i); the Ca(2+)(i) was elevated to 426 +/- 39 nM by 20 microM A1254 but this concentration was not cytotoxic at 1 h. In addition, the mixture, A1254, as well as ortho- and non-ortho-chlorinated PCB congeners (IUPAC Nos. 4, 15, 126, and 138; 5-10 microM) individually decreased GABA(A)-stimulated Ca(2+)(i) responses and this tended not to depend on increases in basal Ca(2+)(i). In cultures DIV 7 and older, A1254 (20 microM) also impaired inhibitory GABA(A) responses as evidenced by an approximately 50% reduction of GABA(A)-stimulated Cl(-) influx (from approximately 6 to 8 mM net accumulation in controls). The results demonstrate that: (1) GABA(A) receptor increases in Ca(2+)(i) and Cl(-)(i) are inhibited by 2-20 microM A1254, regardless of whether the responses are at excitatory or inhibitory stages of development; (2) Ca(2+)(i) homeostasis in cortical cells is disrupted by 10 microM A1254; yet (3) disruption of excitatory GABA(A) responses by A1254 or PCB congeners does not necessarily depend on impaired Ca(2+) homeostasis. These novel observations suggest that GABA(A) receptor responses are a sensitive target for PCB effects in the rat developing nervous system.

Extracellular calcium is required for the polychlorinated biphenyl-induced increase of intracellular free calcium levels in cerebellar granule cell culture.

Recent studies from the laboratory indicate that polychlorinated biphenyl (PCB) congeners can alter signal transduction and calcium homeostasis in neuronal preparations. These effects were more pronounced for the ortho-substituted, non-coplanar congeners, although the mechanisms underlying these effects are not clear. In the present study the time-course and concentration-dependent effects of coplanar and non-coplanar PCBs on intracellular free calcium concentration ([Ca2+]i) in cerebellar granule cell cultures were compared using the fluorescent probe fura-2. The ortho-substituted congeners 2,2'-dichlorobiphenyl (DCB) and 2,2',4,6,6'-pentachlorobiphenyl (PeCB) caused a gradual increase of [Ca2+]i while the non-ortho-substituted congeners 4,4'-DCB and 3,3',4,4',5-PeCB had no effect. The increase of [Ca2+]i produced by 2,2'-DCB was time- and concentration-dependent. Further studies examined possible mechanisms for this rise in [Ca2+]i. In contrast to the muscarinic agonist carbachol, the effects of 2,2'-DCB on [Ca2+]i were not blocked by thapsigargin and required the presence of extracellular calcium. The effects of ortho-substituted PCBs may depend on their ability to inhibit calcium sequestration as 2,2'-DCB significantly inhibited 45Ca2+-uptake by microsomes and mitochondria while 3,3',4,4',5-PeCB had no effect. In addition, 2,2'-DCB significantly increased the binding of [3H]inositol 1,4,5-trisphosphate to receptors on cerebellar microsomes, suggesting another possible mechanism by which ortho-substituted PCBs can mobilize [Ca2+]i. These results show that PCBs increase [Ca2+]i in vitro via a mechanism that requires extracelluar calcium, and support previous structure-activity studies indicating that ortho-substituted PCBs are more potent than non-ortho-substituted PCBs.

Program development and routine notification in a large, independent OPO: a 12-year review.

Since its inception 12 years ago, a large, independent OPO experienced a 631% growth in the number of organ donors. These increases in organ recovery were achieved initially through successful mergers, and later, following the mergers, through focused management, OPO organizational development, and hospital marketing and system development. The cumulative percentage increases beginning in 1987 resulted in the OPO achieving 27.2 donors per million population. In 1996 a system of routine notification of all hospital deaths was implemented and a 24-hour communications center was operated. After 3 full years of routine notification, 73% of all deaths were called to the OPO, resulting in the following increases: total referrals, 691%; organ referrals, 41%; organ donors, 16%; bone donors, 149%; skin donors, 123%; and heart valve donors, 78%. The 16% increase in organ donors was twice the national growth rate and significantly more than the 2% growth experienced by the OPO in the year preceding the implementation of routine notification. The OPO has demonstrated sustained growth over the past decade in a time when erosion of donor recovery levels is always a possibility and frequently a reality for many OPOs.

Ethical analysis of organ recovery denials by medical examiners, coroners, and justices of the peace.

CONTEXT: Despite its pivotal nature, until the early 1990s the role of medical examiners, coroners, and justices of the peace was largely ignored in discussions of the critical shortage of organs for transplantation in the United States. These officials have the right to determine, from a medico-legal perspective, whether a deceased person can be an organ donor. Thus, they play an important role in the donation process. Using a principles-based ethical framework, this article examines the problem of nonrecovery of life-saving organs for transplantation in the United States because a medical examiner or other official denies recovery. OBJECTIVE: The goals of organ donation and the collection of forensic evidence are not mutually exclusive. An analysis of the ethical principles and obligations of beneficence, respect for autonomy, and justice reveals that medical examiners and other officials could probably, after appropriate review, release all cases under their jurisdiction for organ donation. CONCLUSION: Medical examiners, coroners, and justices of the peace could assume a leadership role, working together on public policy with medical, social, and legal groups, spearheading efforts to stop the loss of organs due to official denials, up to and including state and federal regulation and legislation. Beyond their professional obligations, as agents of a social institution, medical examiners and other officials have the more general ethical responsibility of promoting the public health and welfare and of reinforcing societal consensus that transplantation is a social good which should be optimized through formal and informal activities.

Effects of the chlorotriazine herbicide, cyanazine, on GABA(A) receptors in cortical tissue from rat brain.

Chlorotriazine herbicides disrupt luteinizing hormone (LH) release in female rats following in vivo exposure. Although the mechanism of action is unknown, significant evidence suggests that inhibition of LH release by chlorotriazines may be mediated by effects in the central nervous system. GABA(A) receptors are important for neuronal regulation of gonadotropin releasing hormone and LH release. The ability of chlorotriazine herbicides to interact with GABA(A) receptors was examined by measuring their effects on [3H]muscimol, [3H]Ro15-4513 and [35S]tert-butylbicyclophosphorothionate (TBPS) binding to rat cortical membranes. Cyanazine (1-400 microM) inhibited [3H]Ro15-4513 binding with an IC50 of approximately 105 microM (n=4). Atrazine (1-400 microM) also inhibited [3H]Ro15-4513 binding, but was less potent than cyanazine (IC50 = 305 microM). However, the chlorotriazine metabolites diaminochlorotriazine, 2-amino-4-chloro-6-ethylamino-s-triazine and 2-amino-4-chloro-6-isopropylamino-s-triazine were without significant effect on [3H]Ro15-4513 binding. Cyanazine and the other chlorotriazines were without effect on [3H]muscimol or [35S]TBPS binding. To examine whether cyanazine altered GABA(A) receptor function, GABA-stimulated 36Cl- flux into synaptoneurosomes was examined. Cyanazine (50-100 microM) alone did not significantly decrease GABA-stimulated 36Cl- flux. Diazepam (10 microM) and pentobarbital (100 microM) potentiated GABA-stimulated 36Cl- flux to 126 and 166% of control, respectively. At concentrations of 50 and 100 microM, cyanazine decreased potentiation by diazepam to 112 and 97% of control, respectively, and decreased potentiation by pentobarbital to 158 and 137% of control (n = 6). Interestingly, at lower concentrations (5 microM), cyanazine shifted the EC50 for GABA-stimulated 36Cl- flux into synaptoneurosomes from 28.9 to 19.4 microM, respectively (n = 5). These results suggest that cyanazine modulates benzodiazepine, but not the muscimol (GABA receptor site) or TBPS (Cl- channel), binding sites on GABA(A) receptors. Furthermore, at low concentrations, cyanazine may slightly enhance function of GABA(A) receptors, but at higher concentrations, cyanazine antagonizes GABA(A) receptor function and in particular antagonizes the positive modulatory effects of diazepam and pentobarbital.

Effects of Cd2+, Pb2+ and CH3Hg+ on high voltage-activated calcium currents in pheochromocytoma (PC12) cells: potency, reversibility, interactions with extracellular Ca2+ and mechanisms of block.

Effects of the neurotoxic heavy metals Cd2+, Pb2+ and CH3Hg+ on current carried by Ca2+ ions (I(Ca)) through high-voltage activated Ca2+ channels in nerve growth factor (NGF)-differentiated pheochromocytoma (PC12) cells were examined to characterize possible differences in the mechanism of action of these metals on Ca2+ channel function. Specifically, the potency and reversibility of effect on I(Ca) by each metal was examined, as well as the relationship between extracellular [Ca2+] and potency of block of I(Ca) by Cd2+ and Pb2+. In addition, the effect of each of these metals on Ca2+ channels when applied to the intracellular side of the membrane was also examined. When extracellular solution contained 20, 10 or 5 mM Ca2+, the estimated IC50 values (total metal concentration) for block of I(Ca) were 15, 10, and 6.5 microM for Cd2+ and 7.5, 2.0 and 1.1 microM for Pb2+, respectively. CH3Hg+ (1-10 microM) blocked I(Ca) (20 mM Ca2+) in a time- and concentration-dependent manner. When cells were washed with metal-free solutions, block of I(Ca) by Cd2+ was reversed rapidly, whereas block by Pb2+ was reversed only partially, and block of I(Ca) by CH3Hg+ was not reversed. When Pb2+ and CH3Hg+ treated cells were washed in metal-free solutions containing 50 microM D-penicillamine (DPEN), block of I(Ca) by 10 microM Pb2+ was rapidly and completely reversed, whereas, block of I(Ca) by 5 microM CH3Hg+ was not reversed. Higher concentrations (500 microM) of 2,3-dimercapto-1-propane sulfonic acid (DMPS) did reverse partially the block of I(Ca) by 5 and 10 microM CH3Hg+. When Cd2+, Pb2+ or CH3Hg+ was present in the intracellular solution, Ca2+ channel currents were significantly reduced. These results characterize effects of Cd2+ on Ca2+ channels and demonstrate that Cd2+, Pb2+ and CH3Hg+ differ in their actions on Ca2+ channels.

Texas non-donor-hospital project: a program to increase organ donation in community and rural hospitals.

Identifying and recovering donors from community and rural hospitals present a challenge to organ procurement organizations. A study of non-donor hospitals in the United States was undertaken at Johns Hopkins University, which identified 31 hospitals (in one service area) with the facilities to accommodate organ donation, though an organ donor had not been produced in 3 years. The purpose of this study was to determine whether donors could be produced from these hospitals. A large, geographically dispersed OPO initiated a program consisting of (1) in-house coordinators, and (2) routine notification of all hospital deaths. Following implementation of this program, organ donation increased 387% among the targeted 25 hospitals. The number of hospitals producing at least 1 organ donor increased 133%. The number of organs recovered in the project increased 449%. In-house coordinators, by identifying potential donors and facilitating an organ donor awareness program, can increase the number of organ donors in hospitals with low, but real, donor potential.

Effects of gestational methylmercury exposure on immunoreactivity of specific isoforms of PKC and enzyme activity during post-natal development of the rat brain.

Protein kinase C (PKC)-mediated phosphorylation has been implicated in neuronal growth and differentiation [R.S. Turner, R.L. Mazzei, G.J. Raynor, P.R. Girard, J.F. Kuo, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 3143-3147.]. We examined effects of gestational exposure to the neurotoxicant, methylmercury (CH3Hg), on the developmental profile of immunoreactivity (IR) for alpha, beta, gamma and epsilon PKC isoforms and cytosolic PKC activity. Long-Evans dams were dosed on gestational days (GD)6-15 (p.o.) with 0, 1, or 2 mg kg-1 day-1 CH3Hg dissolved in saline. Pups were sacrificed and perfused with buffered paraformaldehyde on post-natal days (PND) 1, 4, 10, 21, 45 and 85. The brains were sectioned sagittally, stained immunohistochemically, and examined throughout the medial to lateral extent. IR in neuronal cell bodies for PKC isoforms alpha, beta, gamma, and epsilon was densest in the olfactory bulb, hippocampus, shell of the inferior colliculus, pons, cerebral, piriform, and cerebellar cortex, whereas axonal staining was prominent in the brainstem, internal capsule, corpus callosum, anterior commissure, fornix and olfactory tract. In controls, the PKC alpha and epsilon IR was highest on PND1-4, decreased dramatically by PND10, and decreased further by PND21. In the neonate, the regional and cellular distributions of alpha and epsilon IR were similar. The PKC gamma IR was greater at post-weaning ages (PND21-85) with the greatest regional density apparent in the hippocampus, cortex, and cerebellum. Only the highest dose of CH3Hg (2 mg kg-1 day-1; GD6-15) produced a persistent decrease in regional alpha and epsilon, but not beta or gamma IR during the post-natal period. These regional and time-dependent changes in PKC isoforms were complemented by the examination of PKC activity in cortex, olfactory bulb, cerebellum and brainstem. Cytosolic PKC activity increased from PND1 to 10 in cortex, olfactory bulb, and cerebellum. On PND21, PKC activity decreased in the cortex and olfactory bulb, but remained high in the cerebellum. By contrast, PKC activity in the brainstem was highest on PND1 and 4 and decreased dramatically by PND21. CH3Hg (2 mg kg-1 day-1) significantly decreased PKC activity on PND1 and 4 in the cortex. The present results characterize the cellular and regional ontogeny of PKC isoenzymes alpha, beta, gamma and epsilon, and indicate that developmental exposure to CH3Hg can alter the ontogeny of specific isoforms and regional PKC activity.

Repeated exposure of adult rats to Aroclor 1254 causes brain region-specific changes in intracellular Ca2+ buffering and protein kinase C activity in the absence of changes in tyrosine hydroxylase.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants, some of which may be neurotoxic. In vitro studies from this laboratory indicated that noncoplanar PCBs perturbed intracellular signal transduction mechanisms including Ca2+ homeostasis, receptor-mediated inositol phosphate production, and translocation of protein kinase C (PKC). In the present study, we examined the effects of PCBs in vivo by dosing adult male Long-Evans rats orally with Aroclor 1254 (0, 10, or 30 mg/kg/day; 5 days/week for 4 weeks) in corn oil. At 24 h after the last dose, rats were tested for motor activity in a photocell device for 30 min. Immediately, the rats were euthanized, blood was collected for thyroid hormone analysis, and brains were removed, dissected into regions (cerebellum, frontal cortex, and striatum), and subcellular fractions were obtained for neurochemical analysis. Following Aroclor 1254 treatment, body weight gain in the high-dose group was significantly lower than the control and low-dose groups. Horizontal motor activity was significantly lower in rats dosed with 30 mg/kg Aroclor 1254. Ca2+ buffering by microsomes was significantly lower in all three brain regions from the 30 mg/kg group. In the same dose group, mitochondrial Ca2+ buffering was affected in cerebellum but not in cortex or striatum. Similarly, total cerebellar PKC activity was decreased significantly while membrane-bound PKC activity was significantly elevated at 10 and 30 mg/kg. PKC activity was not altered either in cortex or the striatum. Neurotransmitter levels in striatum or cortex were slightly altered in PCB-exposed rats compared to controls. Furthermore, repeated oral administration of Aroclor 1254 to rats did not significantly alter forebrain tyrosine hydroxylase immunoreactivity or enzymatic activity. Circulating T4 (total and free) concentrations were severely depressed at both doses in Aroclor 1254-exposed rats compared to control rats, suggesting a severe hypothyroid state. These results indicate that (1) in vivo exposure to a PCB mixture can produce changes in second messenger systems that are similar to those observed after in vitro exposure of neuronal cell cultures; (2) second messenger systems seem to be more sensitive than alterations in neurotransmitter levels or tyrosine hydroxylase involved in dopamine synthesis during repeated exposure to PCBs; and (3) the observed motor activity changes were independent of changes in striatal dopamine levels.