In silico methods for predicting functional synonymous variants.

Single nucleotide variants (SNVs) contribute to human genomic diversity. Synonymous SNVs are previously considered to be "silent," but mounting evidence has revealed that these variants can cause RNA and protein changes and are implicated in over 85 human diseases and cancers. Recent improvements in computational platforms have led to the development of numerous machine-learning tools, which can be used to advance synonymous SNV research. In this review, we discuss tools that should be used to investigate synonymous variants. We provide supportive examples from seminal studies that demonstrate how these tools have driven new discoveries of functional synonymous SNVs.

Analysis of 3.5 million SARS-CoV-2 sequences reveals unique mutational trends with consistent nucleotide and codon frequencies.

BACKGROUND: Since the onset of the SARS-CoV-2 pandemic, bioinformatic analyses have been performed to understand the nucleotide and synonymous codon usage features and mutational patterns of the virus. However, comparatively few have attempted to perform such analyses on a considerably large cohort of viral genomes while organizing the plethora of available sequence data for a month-by-month analysis to observe changes over time. Here, we aimed to perform sequence composition and mutation analysis of SARS-CoV-2, separating sequences by gene, clade, and timepoints, and contrast the mutational profile of SARS-CoV-2 to other comparable RNA viruses. METHODS: Using a cleaned, filtered, and pre-aligned dataset of over 3.5 million sequences downloaded from the GISAID database, we computed nucleotide and codon usage statistics, including calculation of relative synonymous codon usage values. We then calculated codon adaptation index (CAI) changes and a nonsynonymous/synonymous mutation ratio (dN/dS) over time for our dataset. Finally, we compiled information on the types of mutations occurring for SARS-CoV-2 and other comparable RNA viruses, and generated heatmaps showing codon and nucleotide composition at high entropy positions along the Spike sequence. RESULTS: We show that nucleotide and codon usage metrics remain relatively consistent over the 32-month span, though there are significant differences between clades within each gene at various timepoints. CAI and dN/dS values vary substantially between different timepoints and different genes, with Spike gene on average showing both the highest CAI and dN/dS values. Mutational analysis showed that SARS-CoV-2 Spike has a higher proportion of nonsynonymous mutations than analogous genes in other RNA viruses, with nonsynonymous mutations outnumbering synonymous ones by up to 20:1. However, at several specific positions, synonymous mutations were overwhelmingly predominant. CONCLUSIONS: Our multifaceted analysis covering both the composition and mutation signature of SARS-CoV-2 gives valuable insight into the nucleotide frequency and codon usage heterogeneity of SARS-CoV-2 over time, and its unique mutational profile compared to other RNA viruses.

Synonymous ADAMTS13 variants impact molecular characteristics and contribute to variability in active protein abundance.

The effects of synonymous single nucleotide variants (sSNVs) are often neglected because they do not alter protein primary structure. Nevertheless, there is growing evidence that synonymous variations may affect messenger RNA (mRNA) expression and protein conformation and activity, which may lead to protein deficiency and disease manifestations. Because there are >21 million possible sSNVs affecting the human genome, it is not feasible to experimentally validate the effect of each sSNV. Here, we report a comprehensive series of in silico analyses assessing sSNV impact on a specific gene. ADAMTS13 was chosen as a model for its large size, many previously reported sSNVs, and associated coagulopathy thrombotic thrombocytopenic purpura. Using various prediction tools of biomolecular characteristics, we evaluated all ADAMTS13 sSNVs registered in the National Center for Biotechnology Information database of single nucleotide polymorphisms, including 357 neutral sSNVs and 19 sSNVs identified in patients with thrombotic thrombocytopenic purpura. We showed that some sSNVs change mRNA-folding energy/stability, disrupt mRNA splicing, disturb microRNA-binding sites, and alter synonymous codon or codon pair usage. Our findings highlight the importance of considering sSNVs when assessing the complex effects of ADAMTS13 alleles, and our approach provides a generalizable framework to characterize sSNV impact in other genes and diseases.

Synonymous Variants: Necessary Nuance in Our Understanding of Cancer Drivers and Treatment Outcomes.

Once called "silent mutations" and assumed to have no effect on protein structure and function, synonymous variants are now recognized to be drivers for some cancers. There have been significant advances in our understanding of the numerous mechanisms by which synonymous single nucleotide variants (sSNVs) can affect protein structure and function by affecting pre-mRNA splicing, mRNA expression, stability, folding, micro-RNA binding, translation kinetics, and co-translational folding. This review highlights the need for considering sSNVs in cancer biology to gain a better understanding of the genetic determinants of human cancers and to improve their diagnosis and treatment. We surveyed the literature for reports of sSNVs in cancer and found numerous studies on the consequences of sSNVs on gene function with supporting in vitro evidence. We also found reports of sSNVs that have statistically significant associations with specific cancer types but for which in vitro studies are lacking to support the reported associations. Additionally, we found reports of germline and somatic sSNVs that were observed in numerous clinical studies and for which in silico analysis predicts possible effects on gene function. We provide a review of these investigations and discuss necessary future studies to elucidate the mechanisms by which sSNVs disrupt protein function and play a role in tumorigeneses, cancer progression, and treatment efficacy. As splicing dysregulation is one of the most well-recognized mechanisms by which sSNVs impact protein function, we also include our own in silico analysis for predicting which sSNVs may disrupt pre-mRNA splicing.

Distinct signatures of codon and codon pair usage in 32 primary tumor types in the novel database CancerCoCoPUTs for cancer-specific codon usage.

BACKGROUND: Gene expression is highly variable across tissues of multi-cellular organisms, influencing the codon usage of the tissue-specific transcriptome. Cancer disrupts the gene expression pattern of healthy tissue resulting in altered codon usage preferences. The topic of codon usage changes as they relate to codon demand, and tRNA supply in cancer is of growing interest. METHODS: We analyzed transcriptome-weighted codon and codon pair usage based on The Cancer Genome Atlas (TCGA) RNA-seq data from 6427 solid tumor samples and 632 normal tissue samples. This dataset represents 32 cancer types affecting 11 distinct tissues. Our analysis focused on tissues that give rise to multiple solid tumor types and cancer types that are present in multiple tissues. RESULTS: We identified distinct patterns of synonymous codon usage changes for different cancer types affecting the same tissue. For example, a substantial increase in GGT-glycine was observed in invasive ductal carcinoma (IDC), invasive lobular carcinoma (ILC), and mixed invasive ductal and lobular carcinoma (IDLC) of the breast. Change in synonymous codon preference favoring GGT correlated with change in synonymous codon preference against GGC in IDC and IDLC, but not in ILC. Furthermore, we examined the codon usage changes between paired healthy/tumor tissue from the same patient. Using clinical data from TCGA, we conducted a survival analysis of patients based on the degree of change between healthy and tumor-specific codon usage, revealing an association between larger changes and increased mortality. We have also created a database that contains cancer-specific codon and codon pair usage data for cancer types derived from TCGA, which represents a comprehensive tool for codon-usage-oriented cancer research. CONCLUSIONS: Based on data from TCGA, we have highlighted tumor type-specific signatures of codon and codon pair usage. Paired data revealed variable changes to codon usage patterns, which must be considered when designing personalized cancer treatments. The associated database, CancerCoCoPUTs, represents a comprehensive resource for codon and codon pair usage in cancer and is available at https://dnahive.fda.gov/review/cancercocoputs/ . These findings are important to understand the relationship between tRNA supply and codon demand in cancer states and could help guide the development of new cancer therapeutics.

Academic Pharmacy: Where is Our Influence?

Objective. To evaluate the talents of fellows from cohorts 1-10 of the Academic Leadership Fellows Program (ALFP). Methods. This was a descriptive analysis of previously collected ALFP cohort data reflecting the talents using the Clifton StrengthsFinder assessment tool. Data consisted of 295 fellows from the first 10 years of the ALFP program. The Clifton StrengthsFinder talents were aggregated and analyzed to determine talents (strengths) distribution and domain. The aggregate of the four domains were compared among ALFP fellows using a chi-square analysis with an a priori alpha of .05. Results. Lowest frequency of talents was found in the influencing domain (11.2%), while the domains with the largest frequency of talents were strategic thinking (34.4%) and executing (31.1%). When looking at the specific talents within the domains among the ALFP fellows, achiever (in the executing domain) and learner (in the strategic thinking domain) were the most frequent talents, while command (in the influencing domain) and adaptability (in the relationship building domain) were the least frequent talents. Conclusions. Since the profession is deficient in the influencing and relationship building domains (command and adaptability talents, respectively), this could help explain our slow progress in moving the profession from a product-focused role to a provider-based role. Perhaps the profession should be using a strategy better aligned with our signature leadership domains of executing and strategic thinking and focus on being a member of the health care team by aligning with team-based care rather than obtaining provider status.

Ischemic stroke injury is mediated by aberrant Cdk5.

Ischemic stroke is one of the leading causes of morbidity and mortality. Treatment options are limited and only a minority of patients receive acute interventions. Understanding the mechanisms that mediate neuronal injury and death may identify targets for neuroprotective treatments. Here we show that the aberrant activity of the protein kinase Cdk5 is a principal cause of neuronal death in rodents during stroke. Ischemia induced either by embolic middle cerebral artery occlusion (MCAO) in vivo or by oxygen and glucose deprivation in brain slices caused calpain-dependent conversion of the Cdk5-activating cofactor p35 to p25. Inhibition of aberrant Cdk5 during ischemia protected dopamine neurotransmission, maintained field potentials, and blocked excitotoxicity. Furthermore, pharmacological inhibition or conditional knock-out (CKO) of Cdk5 prevented neuronal death in response to ischemia. Moreover, Cdk5 CKO dramatically reduced infarctions following MCAO. Thus, targeting aberrant Cdk5 activity may serve as an effective treatment for stroke.

The role of Cdk5 in neuroendocrine thyroid cancer.

Medullary thyroid carcinoma (MTC) is a neuroendocrine cancer that originates from calcitonin-secreting parafollicular cells, or C cells. We found that Cdk5 and its cofactors p35 and p25 are highly expressed in human MTC and that Cdk5 activity promotes MTC proliferation. A conditional MTC mouse model was generated and corroborated the role of aberrant Cdk5 activation in MTC. C cell-specific overexpression of p25 caused rapid C cell hyperplasia leading to lethal MTC, which was arrested by repressing p25 overexpression. A comparative phosphoproteomic screen between proliferating and arrested MTC identified the retinoblastoma protein (Rb) as a crucial Cdk5 downstream target. Prevention of Rb phosphorylation at Ser807/Ser811 attenuated MTC proliferation. These findings implicate Cdk5 signaling via Rb as critical to MTC tumorigenesis and progression.

Pulmonary surfactant protein A and surfactant lipids upregulate IRAK-M, a negative regulator of TLR-mediated inflammation in human macrophages.

Alveolar macrophages (AMs) are exposed to frequent challenges from inhaled particulates and microbes and function as a first line of defense with a highly regulated immune response because of their unique biology as prototypic alternatively activated macrophages. Lung collectins, particularly surfactant protein A (SP-A), contribute to this activation state by fine-tuning the macrophage inflammatory response. During short-term (10 min-2 h) exposure, SP-A's regulation of human macrophage responses occurs through decreased activity of kinases required for proinflammatory cytokine production. However, AMs are continuously exposed to surfactant, and the biochemical pathways underlying long-term reduction of proinflammatory cytokine activity are not known. We investigated the molecular mechanism(s) underlying SP-A- and surfactant lipid-mediated suppression of proinflammatory cytokine production in response to Toll-like receptor (TLR) 4 (TLR4) activation over longer time periods. We found that exposure of human macrophages to SP-A for 6-24 h upregulates expression of IL-1 receptor-associated kinase M (IRAK-M), a negative regulator of TLR-mediated NF-κB activation. Exposure to Survanta, a natural bovine lung extract lacking SP-A, also enhances IRAK-M expression, but at lower magnitude and for a shorter duration than SP-A. Surfactant-mediated upregulation of IRAK-M in macrophages suppresses TLR4-mediated TNF-α and IL-6 production in response to LPS, and IRAK-M knockdown by small interfering RNA reverses this suppression. In contrast to TNF-α and IL-6, the surfactant components upregulate LPS-mediated immunoregulatory IL-10 production, an effect reversed by IRAK-M knockdown. In conclusion, these data identify an important signaling regulator in human macrophages that is used by surfactant to control the long-term alveolar inflammatory response, i.e., enhanced IRAK-M activity.

Retreatment of patients nonresponsive to pegylated interferon and ribavirin with daily high-dose consensus interferon.

Background. Current treatment of chronic hepatitis C with pegylated interferon and ribavirin has the ability to eliminate viral infection in about half of the patients treated. Therapeutic options, for those with remaining chronic hepatitis, will remain limited until novel antivirals become available in the future. Consensus interferon is currently available and has demonstrated clinical efficacy with superior invitro antiviral activity, but the maximum tolerated dose is not defined. Methods. We assessed the efficacy of daily high-dose (24 ug) consensus interferon with weight-based (1000-1200 mg daily) ribavirin in HCV genotype 1-infected non-responder patients. Results. Six adverse events were documented in five patients, and the trial was terminated with no subject achieving viral clearance. Conclusions. The occurrence of serious adverse events effectively defined the upper limit of acceptable dose, while also revealing that this dose did not offer enhanced sustained viral clearance.

Medication reconciliation at an academic medical center: implementation of a comprehensive program from admission to discharge.

PURPOSE: The implementation of a comprehensive medication reconciliation program to reduce errors in admission and discharge medication orders at an academic medical center is described. SUMMARY: A multidisciplinary team was formed to assess the current process of obtaining medication histories and to develop a new workflow for the pharmacist to obtain and reconcile medication histories. Pharmacists received intensive training on the new workflow, policies, and procedures. Hospitalwide multidisciplinary education was provided, and the new process was introduced in November 2005. Every inpatient admitted to the hospital has a complete and comprehensive home medication history interview conducted by a pharmacist or designee (pharmacy student or intern with subsequent verification by a pharmacist) within 24 hours of arrival. All components of the medication history are documented utilizing an integrated electronic medical record (EMR) medication documentation tool. Development of the discharge medication reconciliation program began in fall 2006. A discharge medication reconciliation report form was created through the EMR to improve the accuracy of the discharge medication orders. The form provides physicians with complete, accurate medication information and decreases the risk for transcription errors. Finally, a discharge medication report was developed for patients to take home. Analysis of the discharge reconciliation process revealed that medication errors were reduced from 90% to 47% on the surgical unit (95% confidence interval [CI], 42-53%; p = 0.000) and from 57% to 33% on the medicine unit (95% CI, 28-38%; p = 0.000). CONCLUSION: A pharmacy-driven multidisciplinary admission history and medication reconciliation process has reduced medication errors in an academic medical center.

Striatal dysregulation of Cdk5 alters locomotor responses to cocaine, motor learning, and dendritic morphology.

Motor learning and neuro-adaptations to drugs of abuse rely upon neuronal signaling in the striatum. Cyclin-dependent kinase 5 (Cdk5) regulates striatal dopamine neurotransmission and behavioral responses to cocaine. Although the role for Cdk5 in neurodegeneration in the cortex and hippocampus and in hippocampal-dependent learning has been demonstrated, its dysregulation in the striatum has not been examined. Here we show that strong activation of striatal NMDA receptors produced p25, the truncated form of the Cdk5 co-activator p35. Furthermore, inducible overexpression of p25 in the striatum prevented locomotor sensitization to cocaine and attenuated motor coordination and learning. This corresponded with reduced dendritic spine density, increased neuro-inflammation, altered dopamine signaling, and shifted Cdk5 specificity with regard to physiological and aberrant substrates, but no apparent loss of striatal neurons. Thus, dysregulation of Cdk5 dramatically affects striatal-dependent brain function and may be relevant to non-neurodegenerative disorders involving dopamine neurotransmission.

Pyrethroid modulation of spontaneous neuronal excitability and neurotransmission in hippocampal neurons in culture.

Pyrethroid insecticides have potent actions on voltage-gated sodium channels (VGSC), inhibiting inactivation and increasing channel open times. These are thought to underlie, at least in part, the clinical symptoms of pyrethroid intoxication. However, disruption of neuronal activity at higher levels of organization is less well understood. In order to characterize pyrethroid effects on neurotransmitter release and neuronal excitability in glutamatergic networks, we examined the effects of deltamethrin (DM) and permethrin (PM) on neuronal activity in hippocampal neuronal cultures using patch-clamp and microelectrode array (MEA) recordings. In the presence of inhibitors of GABA receptors, spontaneous excitatory post-synaptic currents (sEPSCs) and spontaneous spike rates were reduced in a concentration-dependent manner by both DM and PM. IC(50) values were 0.037 and 0.70microM for inhibition of sEPSCs and 0.60 and 21.8microM for inhibition of spontaneous spike rate by DM and PM, respectively. Both compounds altered burst activity by decreasing the number of spikes during spontaneous bursting, the number of sEPSCs within a bursting release event and the duration of sEPSC bursts while increasing both the interspike interval and the time between sEPSCs. Exposure of neurons to the VGSC-specific modulator veratridine had effects similar to both DM and PM, while inhibition of voltage-gated calcium channels had no effect on spontaneous spike rates. In the absence of GABA receptor antagonists, both DM and PM increased spontaneous spike rates. Altogether, these data demonstrate that DM and PM disrupt network activity in vitro, largely via a VGSC-dependent mechanism.

Modulation of glutamatergic transmission by sulfated steroids: role in fetal alcohol spectrum disorder.

It is well established that sulfated steroids regulate synaptic transmission by altering the function of postsynaptic neurotransmitter receptors. In recent years, evidence from several laboratories indicates that these agents also regulate glutamatergic synaptic transmission at the presynaptic level in an age-dependent manner. In developing neurons, pregnenolone sulfate (PREGS) increases the probability of glutamate release, as evidenced by an increase in the frequency of AMPA receptor-mediated miniature excitatory postsynaptic currents and a decrease in paired-pulse facilitation. In hippocampal slices from postnatal day 3-5 rats, this effect is mediated by an increase in Ca(2+) levels in the axonal terminal that depends on presynaptic NMDA receptors. This is followed by delayed potentiation of postsynaptic AMPA receptor currents. Importantly, depolarization of postsynaptic neurons, inhibition of hydroxysteroid sulfatase activity and acute exposure to ethanol mimics the effect of exogenous PREGS application. This developmental form of synaptic plasticity cannot be observed in slices from rats older than postnatal day 6, when presynaptic NMDA receptors are no longer expressed in CA1 hippocampal region. Both in the CA1 hippocampal region and the dentate gyrus of more mature rats, PREGS, dehydroepiandrosterone sulfate and hydroxysteroid sulfatase inhibitors increase paired-pulse facilitation, without affecting basal glutamate release probability. This effect depends on activation of sigma(1)-like receptors and G(i/o) and involves a target in the release machinery that is downstream of residual Ca(2+). These presynaptic actions of sulfated steroids could play important roles in physiological processes ranging from synapse maturation to learning and memory, as well as pathophysiological conditions such as fetal alcohol spectrum disorder.

Permethrin, but not deltamethrin, increases spontaneous glutamate release from hippocampal neurons in culture.

Pyrethroid insecticide modulation of the voltage-gated sodium channel (VGSC) is proposed to underlie their effects on neuronal excitability. However, some in vitro evidence indicates that target sites other than VGSCs could contribute to pyrethroid disruption of neuronal activity. VGSC-independent, pyrethroid-induced changes in neurotransmitter release were examined to investigate the possibility that target sites other than VGSCs contribute to pyrethroid effects. Using whole-cell patch clamp recordings, deltamethrin and permethrin effects on glutamate-mediated miniature excitatory postsynaptic currents (mEPSCs) from pyramidal neurons in mixed hippocampal cultures were examined. In the presence of the VGSC antagonist tetrodotoxin, the type I pyrethroid permethrin (10 microM) increased the average frequency of mEPSCs from a basal level of 1.0+/-0.4 to 3.5+/-0.6 Hz, with peak frequency of 9.9+/-1.5 Hz (n=6). Permethrin did not affect the distribution of current amplitudes, indicating that permethrin increased the probability of glutamate release at the presynaptic terminal without effects on postsynaptic responses. Removal of calcium from the extracellular solution following the induction of the permethrin-mediated effect decreased mEPSC frequency (6.8+/-1.8 Hz, n=3) to near control levels (1.9+/-0.8 Hz for control versus 2.5+/-0.6 Hz for permethrin minus Ca(2+), respectively). However, the N- and P/Q-type voltage-gated calcium channel antagonist omega-conotoxin MVIIC had no effect on the permethrin-dependent increase in mEPSC frequency. In contrast to permethrin, the type II pyrethroid deltamethrin (10 microM) failed to affect mEPSC frequency. These results indicate that permethrin causes a calcium-dependent increase in glutamate release from hippocampal neurons that is independent of effects on voltage-gated sodium or N- or P/Q-type voltage-gated calcium channels. The data indicate that permethrin increases mEPSC frequency via an alteration in intracellular calcium dynamics at the presynaptic terminal.

Developmental neurotoxicity of pyrethroid insecticides: critical review and future research needs.

Pyrethroid insecticides have been used for more than 40 years and account for 25% of the worldwide insecticide market. Although their acute neurotoxicity to adults has been well characterized, information regarding the potential developmental neurotoxicity of this class of compounds is limited. There is a large age dependence to the acute toxicity of pyrethroids in which neonatal rats are at least an order of magnitude more sensitive than adults to two pyrethroids. There is no information on age-dependent toxicity for most pyrethroids. In the present review we examine the scientific data related to potential for age-dependent and developmental neurotoxicity of pyrethroids. As a basis for understanding this neurotoxicity, we discuss the heterogeneity and ontogeny of voltage-sensitive sodium channels, a primary neuronal target of pyrethroids. We also summarize 22 studies of the developmental neurotoxicity of pyrethroids and review the strengths and limitations of these studies. These studies examined numerous end points, with changes in motor activity and muscarinic acetylcholine receptor density the most common. Many of the developmental neurotoxicity studies suffer from inadequate study design, problematic statistical analyses, use of formulated products, and/or inadequate controls. These factors confound interpretation of results. To better understand the potential for developmental exposure to pyrethroids to cause neurotoxicity, additional, well-designed and well-executed developmental neurotoxicity studies are needed. These studies should employ state-of-the-science methods to promote a greater understanding of the mode of action of pyrethroids in the developing nervous system.

A retrospective review of liver transplant patients treated with sirolimus from a single center: an analysis of sirolimus-related complications.

BACKGROUND: Sirolimus (SRL) is a powerful immunosuppressant used primarily in calcineurin inhibitors (CNI)-related nephrotoxicity. However, reports of drug-related side effects are increasing. The aim of our report is to review the frequency and timing of these complications within our transplant patient population. METHODS: We retrospectively reviewed the medical records of liver-transplanted patients treated with sirolimus between November 1998 and April 2002. The data collected included SRL serum levels, frequency of reported and documented SRL-related side effects, and survival outcomes. Statistical evaluation included Pearson chi-square and the Fisher's exact tests. RESULTS: Overall, 205 patients were identified, with 30 patients removed from the analysis for different reasons. Of the remaining 175 patients, 91 (52%) patients developed a complication other than an increase in serum triglycerides and/or cholesterol. The most frequent complications were: bilateral lower extremity edema (57.1%), dermatitis (25.3%), oral ulcers (24.2%), joint pain (23.0%), pleural effusion (16.5%) and increase in abdominal girth (9.9%). Other complications included: generalized edema (5.5%), pericardial effusion (5.5%), facial edema (2.2%), and upper extremity edema (1.3%). In addition, we reported two cases of hepatic artery thrombosis, one case of wound dehiscence with evisceration that required surgical repair, and one case of skin cancer. Interestingly, we found that a previous history of myocardial ischemia correlates with the development of SRL side effects. CONCLUSIONS: SRL is a powerful immunosuppressant but not devoid of side effects. These results have elevated our level of suspicion when instituting SRL and may help with early recognition and prevention of drug related complications.

Effects of pyrethroids on voltage-sensitive calcium channels: a critical evaluation of strengths, weaknesses, data needs, and relationship to assessment of cumulative neurotoxicity.

The Food Quality Protection Act of 1996 requires that the U.S. Environmental Protection Agency conduct cumulative risk assessments for classes of pesticides that have a common mode or mechanism of action. For the pyrethroid insecticides, disruption of voltage-sensitive sodium channel function is generally accepted as the mechanism underlying acute neurotoxicity. However, data exist which suggest that voltage-sensitive calcium (Ca(2+)) channels (VSCC) may also be important targets of pyrethroid action. VSCC are important to neuronal function during development and for neurotransmitter release, gene expression, and electrical excitability in the nervous system. Disruption of these and other processes mediated by VSCC can result in neurotoxicity. If effects on VSCC are demonstrated to contribute to pyrethroid neurotoxicity, then such effects will have to be considered when making decisions regarding cumulative risk of exposure to this class of compounds. This document provides a critical review of the data related to the hypothesis that VSCC are important targets of pyrethroid effects. Data supporting effects of pyrethroids on VSCC have been generated by several different laboratories using different techniques and biological preparations. Thus, the many reports of effects on VSCC provide evidence that pyrethroids may interact with VSCC. However, evidence to support a role of VSCC in pyrethroid neurotoxicity is based entirely on in vitro observations, and numerous limitations exist in these data, including: (1) lack of defined concentration-response relationships, with some effects observed only at relatively high concentrations, (2) the use of indirect measures of VSCC function, (3) data from nonmammalian species, (4) data from studies that have not been peer-reviewed, (5) the need for replication of some effects, and (6) inconsistent or contradictory results from different laboratories/preparations. Thus, at the present time, it is premature to conclude that effects on VSCC play an important role in the acute neurotoxicity of pyrethroid insecticides in mammals. To demonstrate that VSCC are important targets of pyrethroid neurotoxicity in mammals, in vivo studies supporting a role for pyrethroid effects on VSCC are needed. Additional support could be provided by demonstration of direct effects of pyrethroid compounds on mammalian neuronal VSCC in vitro, including demonstration that concentration-response relationships are similar, or greater, in sensitivity to effects of pyrethroids on voltage-sensitive sodium channels. If such effects were to be demonstrated, the rationale for considering VSCC as targets of pyrethroid compounds when assessing cumulative risk would be strengthened. However, at the present time, the data available neither support nor refute conclusively the hypothesis that effects on VSCC are important to the acute neurotoxicity of pyrethroids.