Application of risk score analysis to low-coverage whole genome sequencing data for the noninvasive detection of trisomy 21, trisomy 18, and trisomy 13.

OBJECTIVES: Clinical performance of a low coverage, low cost, massively parallel sequencing (MPS)-based assay to stratify risk of trisomy 21, 18, and 13 pregnancies was determined. METHODS: The study included 1100 samples with birth outcome or karyotype results, comprising low-risk patients (84.2%) negative for risk indications from maternal age, serum screening, ultrasound, or family history, and high-risk patients (15.8%) with at least one of the aforementioned indications. Cell free DNA (cfDNA) was extracted from maternal plasma. Library preparation incorporated 96 index barcodes to enable sequencing on a HiSeq 2000 or 2500. Risk scores were calculated using chromosomal representation, fetal fraction, and maternal age at the estimated date of delivery. A risk score greater than or equal to 1 in 100 was used to stratify samples as high risk for trisomy 21, trisomy 18, or trisomy 13. RESULTS: Sensitivity and specificity were calculated based on risk group stratification. Trisomy 21, trisomy 18, and trisomy 13 were detected with greater than 99% sensitivity and 99.9% specificity. Fetal sex classification accuracy was 99.3%. CONCLUSIONS: We conclude that simplified MPS can be used to stratify the risk of pregnancies for trisomy 21, trisomy 18, and trisomy 13 and accurately determine fetal sex. © 2015 John Wiley & Sons, Ltd.

Circulating cell free DNA testing: are some test failures informative?

OBJECTIVE: The proportion of circulating cell free DNA derived from the feto-placental unit (fetal fraction or FF) correlates with test success and interpretation reliability. Some fetal disorders are associated with systematically lower FF, sometimes resulting in noninformative results. METHODS: We analyzed results from pregnancies tested in a nested case/control study derived from a cohort of 4664 high-risk pregnancies. Low FF was defined before and after adjusting for maternal weight and gestational age. RESULTS: Compared with euploid pregnancies, the median FF was significantly higher in Down syndrome pregnancies (ratio 1.17) and significantly lower in trisomy 18 and triploid pregnancies (ratios 0.71 and 0.19, respectively). Among 2157 pregnancies tested, 13 (0.6%) had FF <3.0% (all noninformative), including three trisomy 18 and three triploidy fetuses. After adjustment, 16 pregnancies (0.7%) had FF <0.3 multiples of the median (six informative), including one trisomy 18 and three triploidy fetuses. Modeled positive predictive values for low and high-risk populations were 7% and 30%, respectively. CONCLUSION: Among women with noninformative results attributable to low FF, trisomy 18 and/or triploidy risk are sufficiently high to warrant offering additional assessments (e.g. ultrasound). If the testing indication is ultrasound abnormality, amniocentesis and karyotype/microarray should be considered. © 2014 John Wiley & Sons, Ltd.

Liver enzymes in White Leghorns selected for the sheep red blood cell immune response.

Liver enzymes are essential to xenobiotic metabolism. Expression of these enzymes is dependent upon factors such as age and sex. The objective of this study was to determine basal liver enzyme levels in male and female White Leghorn chickens to provide reference values for future studies. Chickens from 2 lines divergently selected for 35 generations for high antibody and low antibody immune response to SRBC were used. Six male and 6 female chickens from each line were killed at each of 4, 8, 12, and 20 wk of age. Livers were collected and used for enzyme analyses. Liver tissue was analyzed for quinone reductase, glutathione-S-transferase, and cytochrome P450 3A4 activity. All data were analyzed using ANOVA. There were no consistent differences in enzyme activity between high- and low-antibody lines at any age. Cytochrome P450 3A4 activity was substantially greater in 4- and 8-wk than in 12- and 20-wk-old chickens (P < 0.001). This study provides insights into enzyme activities of liver enzymes; however, except for cytochrome P450 3A4, no clear trends across ages were observed.

Characterization of the methylation patterns of MS4A2 in atopic cases and controls.

BACKGROUND: It is largely unknown whether epigenetic modifications of key genes may contribute to the reported maternal effects in atopy. The aim of this study was to characterize the methylation patterns of the membrane-spanning 4-domains, subfamily A, member 2 gene (MS4A2) (beta-chain of the IgE high-affinity receptor), a key gene in the allergic cascade. METHODS: Mass spectrometry and bisulphite sequencing were used to measure the methylation of two potential substrates for epigenetic regulation of MS4A2, namely a predicted promoter and a CpG-rich AluSp repeat. Methylation was measured in DNA extracted from peripheral blood lymphocytes of 38 atopic cases and 37 controls. Cases were positive for atopy, asthma, bronchial hyper-responsiveness and had high IgE levels. Both parents of eight atopic cases were also tested. RESULTS: The AluSp element was highly methylated across all individuals (mean 0.92, range 0.87-0.94), a pattern inconsistent with classical imprinting. Variation in methylation at this locus was not associated with age, sex, daily steroid use or atopic status, and there were no differences in methylation between mothers and fathers of atopic cases. Bisulphite sequencing analysis of the promoter region showed that it was also not imprinted, and there was no evidence for allele-specific methylation, but we were unable to test for association with atopy status. CONCLUSIONS: Methylation levels at the AluSp repeat analysed in MS4A2 were inconsistent with classical imprinting mechanisms and did not associate with atopy status. The promoter region was less methylated but further analysis of this region in larger cohorts is warranted to investigate its role in allergic disease.

Vacuolation of sensory ganglion neuron cytoplasm in rats with long-term exposure to organophosphates.

Cytoplasmic vacuolation of sensory neurons has been reported to occur within the dorsal root ganglia in studies investigating various neuropathic conditions including the effects of neurotoxic chemicals. In this study, we investigated this lesion in adult (98-119 days old) male Long-Evans rats, after multiple exposures to two organophosphates (tri-ortho-tolyl phosphate [TOTP] and chlorpyrifos) and the modifying effects of concurrent corticosterone. Tri-ortho-tolyl phosphate was administered by gavage (75, 150, or 300 mg/kg) every other day between days 14 and 28 and between days 49 and 63, chlorpyrifos (60 mg/kg) was administered subcutaneously on days 7 and 42, and corticosterone was provided in the drinking water throughout the study at a concentration of 400 microg/mL. Although relatively uncommon, there was an increase in frequency of cytoplasmic vacuoles seen in treatment groups having multiple exposures to TOTP. They were characterized as peripherally located, single-limiting membrane-bound structures in the neuronal perikarya. There was no associated cell death, even when vacuoles were large. This is the initial report of an association of this change following exposure to neurotoxic organophosphates.

Effects of silymarin on gossypol toxicosis in divergent lines of chickens.

Gossypol, a pigment of cotton, is a hepatic toxin for chickens. Thus, despite its high protein content, inclusion of cottonseed meal in poultry diets is problematic. Silymarin, an extract from milk thistle, has hepatoprotective qualities and could potentially serve as a feed additive to offset the toxicity of gossypol. The objective of this study was to determine if silymarin could counteract gossypol toxicosis. Cockerels (n = 144) from lines divergently selected for humoral immunity were used. Three individuals from each line were randomly assigned to a cage and fed a corn-soybean meal (control) diet for 14 d. Six cages per line were then randomly assigned 1 of 4 dietary treatments (1,000 mg/kg of gossypol, 1,000 mg/kg of silymarin, 1,000 mg/kg of both gossypol and silymarin, or a control diet). Body weight and feed intake data were collected for 21 d, with chickens bled weekly to collect plasma and determine hematocrits. Chickens were then killed, and livers were collected for subsequent histology and enzymatic activity analyses. Endpoints measured weekly were analyzed with repeated measures and regression methodologies. Plasma and liver enzyme activities, and histological measures, were analyzed using ANOVA. No significant interactions between diets and lines were observed. Chickens assigned to the gossypol and gossypol-silymarin diets stopped gaining weight at d 14 (P < 0.001) and lost weight by d 21 (P < 0.001). Gamma glutamyltransferase was also elevated in these chickens at d 14; activities increased further by d 21 (P < 0.001). Histological examination of liver slices indicated substantial lipidosis (P < 0.001). Furthermore, quinone reductase activity was higher in gossypol- and gossypol-silymarin-treated chickens than in control and silymarin-treated chickens (P < 0.001). Silymarin did not alleviate any clinical effects of gossypol toxicosis.

Characterization of bovine neutrophil beta2-adrenergic receptor function.

This study compares bovine leukocyte beta-adrenergic receptor densities to that of the rat, demonstrates for the first time a functional beta(2)-adrenergic receptor signaling pathway in steer neutrophils, and investigates the effect of an inflammatory stimulus on that signaling pathway. The beta(1)-/beta(2)-adrenergic antagonist ([3H])CGP-12177 demonstrated that rat lymphocyte specific binding-site density was highest, followed by steer and dairy cow lymphocytes, and lastly steer and dairy cow neutrophils. The beta(2)-adrenergic agonist terbutaline stimulated steer neutrophil adenosine 3,5-cyclic monophosphate (cAMP) production, an effect increased by inclusion of > or = 1 x 10(-8) M phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C. Both terbutaline and the nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) independently decreased steer neutrophil superoxide anion production in a concentration-dependent manner, with 1 x 10(-4) M IBMX enhancing both the potency and efficacy of the terbutaline effect (up to 74% reduction in superoxide anion production). Superoxide anion production was also reduced by the synthetic cAMP analog 8-bromo-cAMP, which increased the potency of the IBMX effect on superoxide anion production. Taken together, these data demonstrate the presence of a beta(2)-adrenergic receptor signaling pathway in bovine neutrophils much like that described in other animal species, as well as the potential for an inflammatory stimulus to alter its function.

Downregulation of RUNX3 and TES by hypermethylation in glioblastoma.

Glioblastoma, the most aggressive and least treatable form of malignant glioma, is the most common human brain tumor. Although many regions of allelic loss occur in glioblastomas, relatively few tumor suppressor genes have been found mutated at such loci. To address the possibility that epigenetic alterations are an alternative means of glioblastoma gene inactivation, we coupled pharmacological manipulation of methylation with gene profiling to identify potential methylation-regulated, tumor-related genes. Duplicates of three short-term cultured glioblastomas were exposed to 5 microM 5-aza-dC for 96 h followed by cRNA hybridization to an oligonucleotide microarray (Affymetrix U133A). We based candidate gene selection on bioinformatics, reverse transcription-polymerase chain reaction (RT-PCR), bisulfite sequencing, methylation-specific PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Two genes identified in this manner, RUNX3 and Testin (TES), were subsequently shown to harbor frequent tumor-specific epigenetic alterations in primary glioblastomas. This overall approach therefore provides a powerful means to identify candidate tumor-suppressor genes for subsequent evaluation and may lead to the identification of genes whose epigenetic dysregulation is integral to glioblastoma tumorigenesis.

Neurological effects of acute uranium exposure with and without stress.

Circulating uranium rapidly enters the brain and may cause adverse effects on the nervous system that are potentially modulated by stress. In this study, the neurological effects of a single intramuscular injection of 0, 0.1, 0.3, or 1 mg uranium/kg (as uranyl acetate, UA) in rats were examined in the presence and absence of stress. Treatment with UA produced time and dose-dependent increases in serum and regional brain uranium levels. While serum levels returned to control levels by day 30, brain levels remained elevated. Application of stress did not affect the distribution or retention of uranium. Exposure to 1 mg U/kg significantly decreased ambulatory activity, weight gain, forelimb grip strength and transiently impaired working memory. Effects on grip strength and memory were prevented by application of stress prior to uranium exposure. Striatal dopamine content was reduced by 30% 3 days after treatment with 1mg/kg (59+/-6 nmol/mg tissue versus 41+/-5 nmol/mg tissue), but levels returned to control 7 days after uranium exposure. The effect on dopamine was ameliorated by prior application of stress. Exposure to UA did not alter 3,4 dihydroxyphenylacetic acid (DOPAC) levels or numbers of D2 receptors in the striatum. No effect of uranium or stress was observed on levels of GABA, serotonin, norepinephrine, or glutathione (GSH) in the striatum, hippocampus, cerebellum, or cortex. These results indicate that single intramuscular exposures to uranium produce sustained elevation of brain uranium levels and at doses above 0.3 mg/kg can have adverse neurological effects. Application of stress prior to uranium administration modulates neurological effects, but the mechanism is not due to effects on uranium distribution. Uranium exposure also produced renal toxicity which must be considered to accurately assess the effects of uranium on neurological function.

Early effects of neuropathy-inducing organophosphates on in vivo concentrations of three neurotrophins.

Exposure to OP compounds that inhibit neurotoxic esterase (NTE) induces a delayed neuropathy (OPIDN) characterized by Wallerian-like degeneration of long axons in certain animals, including humans. Pope et al. (Toxicol. Lett. 75:111-117, 1995) found that neurite outgrowth occurred following the addition of spinal cord extracts from chickens with active OPIDN to neuroblastoma cells, suggesting growth factor expression during the neuropathy. We hypothesized that, shortly after exposure to a neuropathic OP compound, the central nervous system (CNS) attempts to recover from the toxic insult through upregulation of the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) in susceptible regions of the nervous system. We hypothesized that such upregulation is transient and cannot be sustained. To test this hypothesis, we exposed 10-week-old chickens to a neuropathic OP compound (PSP, 2.5 mg/kg), a non-neuropathic OP compound (paraoxon, 0.10 mg/kg), and vehicle (DMSO, 0.5 ml/kg) intramuscularly. By day 8, all PSP-treated birds demonstrated clinical signs of OPIDN. We sacrificed chickens by pentobarbital overdose at 4, 8, 24, and 48 hours, and 5 and 10 days post-exposure and confirmed NTE inhibition in birds treated with PSP 4 and 24 hours earlier. Enzyme-linked immunosorbant assays indicated that NGF, BDNF, and NT-3 are found in chicken lumbar spinal cord after exposure to a neuropathic OP compound. However, exposure to the neuropathic OP compound, PSP, did not preferentially elevate levels of NGF, BDNF, and NTE compared to the non-neuropathic OP compound, paraoxon. This suggests that these neurotrophins alone do not contribute to a sustained regenerative effort in the CNS.

Effects of organophosphorus compounds on ATP production and mitochondrial integrity in cultured cells.

Recent studies in vivo and in vitro suggested that mitochondrial dysfunction follows exposure to organophosphorus (OP) esters. As mitochondrial ATP production is important for cellular integrity, ATP production in the presence of OP neurotoxicants was examined in a human neuronal cell line (SH-SY5Y neuroblastoma cells) and primary dorsal root ganglia (DRG) cells isolated from chick embryos and subsequently cultured to achieve maturation with axons. These cell culture systems were chosen to evaluate toxic effects on the mitochondrial respiratory chain associated with exposure to OP compounds that do and do not cause OP-induced delayed neuropathy (OPIDN), a disorder preceded by inhibition of neurotoxic esterase (NTE). Concentration- and time-response studies were done in neuroblastoma cells exposed to phenyl saligenin phosphate (PSP) and mipafox, both compounds that readily induce delayed neuropathy in hens, or paraoxon, which does not. Phenylmethylsulfonyl fluoride (PMSF) was included as a non-neuropathic inhibitor of NTE. Purified neuronal cultures from 9 day-old chick embryo DRG were treated for 12 h with 1 microM PSP, mipafox, or paraoxon. In situ evaluation of ATP production measured by bioluminescence assay demonstrated decreased ATP concentrations both in neuroblastoma cells and chick DRG neurons treated with PSP. Mipafox decreased ATP production in DRG but not in SH-SY5Y cells. This low energy state was present at several levels of the mitochondrial respiratory chain, including Complexes I, II, III, and IV, although Complex I was the most severely affected. Paraoxon and PMSF were not effective at all complexes, and, when effective, required higher concentrations than needed for PSP. Results suggest that mitochondria are an important early target for OP compounds, with exposure resulting in depletion of ATP production. The targeting of neuronal, rather than Schwann cell mitochondria in DRG following exposure to PSP and mipafox was verified by loss of the mitochondrial-specific dye, tetramethylrhodamine, in these cells. No such loss was seen in paraoxon exposed neurons isolated from DRG or in Schwann cells treated with any of the test compounds.

Comparison of two in vitro activation systems for protoxicant organophosphorous esterase inhibitors.

In order to perform in vitro testing of esterase inhibition caused by organophosphorous (OP) protoxicants, simple, reliable methods are needed to convert protoxicants to their esterase-inhibiting forms. Incubation of parathion or chlorpyrifos with 0.05% bromine solution or uninduced rat liver microsomes (RLM) resulted in production of the corresponding oxygen analogs of these OP compounds and markedly increased esterase inhibition in SH-SY5Y human neuroblastoma cells. Neither activation system affected cell viability or the activity of AChE or NTE in the absence of OP compounds. Although parathion and chlorpyrifos were activated by RLM, bromine activation required fewer steps and produced more esterase inhibition for a given concentration of chlorpyrifos. However, RLM activation of OP protoxicants produced metabolites other than oxygen analogs and may, therefore, be more relevant as a surrogate for OP biotransformation in vivo. This methodology makes the use of intact cells for in vitro testing of esterase inhibition caused by protoxicant organophosphate compounds a viable alternative to in vivo tests.

Bridging the gap between in vitro and in vivo models for neurotoxicology.

In vitro systems are widely used for investigation of neurotoxicant-induced perturbations of cellular functions. A variety of systems exist that demonstrate certain similarities to neurotoxicant-induced events in the intact animal are discussed, including single-cell types, systems that consider endpoints relevant in toxicology, and systems that consider heterogeneous cell interactions. Relationships between the in vitro and in vivo systems are examined in which ethanol, lead, polychlorinated biphenyl compounds, and organophosphate insecticides are examples. Situations in which the in vitro systems have been used to advantage are provided, along with cautions associated with their use.

Common mechanism of toxicity: a case study of organophosphorus pesticides.

The Food Quality Protection Act of 1996 (FQPA) requires the EPA to consider "available information concerning the cumulative effects of such residues and other substances that have a common mechanism of toxicity ... in establishing, modifying, leaving in effect, or revoking a tolerance for a pesticide chemical residue." This directive raises a number of scientific questions to be answered before the FQPA can be implemented. Among these questions is: What constitutes a common mechanism of toxicity? The ILSI Risk Science Institute (RSI) convened a group of experts to examine this and other scientific questions using the organophosphorus (OP) pesticides as the case study. OP pesticides share some characteristics attributed to compounds that act by a common mechanism, but produce a variety of clinical signs of toxicity not identical for all OP pesticides. The Working Group generated a testable hypothesis, anticholinesterase OP pesticides act by a common mechanism of toxicity, and generated alternative hypotheses that, if true, would cause rejection of the initial hypothesis and provide criteria for subgrouping OP compounds. Some of the alternative hypotheses were rejected outright and the rest were not supported by adequate data. The Working Group concluded that OP pesticides act by a common mechanism of toxicity if they inhibit acetylcholinesterase by phosphorylation and elicit any spectrum of cholinergic effects. An approach similar to that developed for OP pesticides could be used to determine if other classes or groups of pesticides that share structural and toxicological characteristics act by a common mechanism of toxicity or by distinct mechanisms.

Interaction of Clostridium difficile toxins and mouse hepatic microsomes.

Intraperitoneal administration of toxins of Clostridium difficile to mice resulted in loss of hepatic cytochrome P450 and peroxidation of microsomal lipids. Pretreatment with the microsomal enzyme inducer beta-naphthoflavone partially alleviated these effects and increased survival time of intoxicated animals.

Biochemical and pathological effects of Clostridium difficile toxins in mice.

Toxins produced by Clostridium difficile are lethal to mice after i.p. administration. Among the alterations observed when mice were given a preparation containing both Toxin A and Toxin B were a 1.6 +/- 0.2 degrees C (mean +/- S.E., N = 7) depression of rectal body temperature, blood in the liver (318 +/- 13% of control levels) and a decrease in glutathione concentration (74 +/- 2% of control). Purified Toxin A and purified Toxin B were both able to alter these parameters. Toxin B, however, had a more profound effect on serum isocitrate dehydrogenase levels (raised to 198 +/- 18% of control) and liver O-demethylase activity (reduced to 64 +/- 8% of control), parameters sensitive to alteration in liver damage. The effects of Toxin B on these parameters were partially alleviated in mice pretreated with N-acetylcysteine (1.2 g/kg i.p.) and triamcinolone (120 mg/kg i.p.) and, although the percentage of survivors did not improve, survival time was increased from 3.0 +/- 0.1 hr to 4.6 +/- 0.5 and 5.7 +/- 1.3 hr, respectively, by these agents.

Evaluation of knit glove fabrics as barriers to dermal absorption of organophosphorus insecticides using an in vitro test system.

Cotton and synthetic knit glove fabrics in combination with an in vitro skin model were used to examine the capability of fabric to decrease the dermal absorption of the organophosphorus insecticides azinphos-methyl, paraoxon, and malathion. Capability for inhibition of acetylcholinesterase was determined in samples of media taken from under the skin barrier after the skin model, with or without fabric protection, had been exposed to the test compounds for 4 h. Acetylcholinesterase inhibitions caused by the direct addition of organophosphorus insecticide to the media were also included in the comparison. Results indicated that the skin model system alone had some capability to serve as a barrier to the transfer of organophosphates. Fabric covering used on the test model increased the barrier between insecticide application and resultant acetylcholinesterase inhibition. The all-cotton, 7-cut knit was especially effective in preventing the absorption of azinphos-methyl, as this organophosphorus insecticide had no capability to cause acetylcholinesterase inhibition when this fabric was used to protect the skin model. Knit glove materials of 100% cotton were demonstrated to be effective in preventing the absorption of paraoxon and malathion. These studies indicate that an in vitro model system can be used in combination with fabrics to study the relationship between clothing and skin as barriers to the absorption of organophosphorus insecticides.

Increase in glucuronide conjugation of aflatoxin P1 after pretreatment with microsomal enzyme inducers.

Microsomes prepared from livers of chickens given enzyme inducers had increased capability to convert aflatoxin P1 to its glucuronide conjugate. This capability was 190% +/- 19 and 184% +/- 13 of control values (mean +/- S.E., N = 4) 96 h after intraperitoneal administration of 80 mg/kg beta-naphthoflavone or 3-methylcholanthrene, respectively. Glucuronidation of aflatoxin P1 was also increased 15 days after 500 mg/kg i.p. of a polychlorinated biphenyl mixture (to 471% +/- 111). Fifteen days on a low protein diet (containing 54% of normal levels) did not alter aflatoxin glucuronidation. Increased glucuronidation after administration of inducers was due to increased specific activity of the microsomal enzymes.

Alterations of cytoskeletal tau protein of SH-SY5Y human neuroblastoma cells after exposure to MPTP.

In this study, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 10(-3) to 10(-4) M for 2 to 5 days) increased the expression of microtubule-associated tau protein in both the supernatant and pellet fractions of lysed SH-SY5Y human neuroblastoma cells. The western blot using anti-tau-1 antibodies demonstrated that the cells contained at least six isoforms of tau proteins, five with molecular weights from 45 to 62 kD. Reverse transcriptase polymerase chain reaction (RT-PCR) using primers coding whole length tau protein further confirmed the presence of tau in SH-SY5Y cells. The PCR product of tau in SH-SY5Y cells had approximately 1050 base pairs. MPTP caused an increased expression of the PCR product of tau, suggesting that the toxicant caused an increase in mRNA coding the tau protein. The expression of cytoskeletal tau protein may, therefore, provide a marker for MPTP neurotoxicity in SH-SY5Y cells.

Organophosphorus compounds alter intracellular F-actin content in SH-SY5Y human neuroblastoma cells.

Cytoskeletal components, especially f-actin (filamentous actin), are responsible for neurite extension and maintenance. Alterations in neurite length and quality precede in vitro cell death induced by organophosphorus (OP) compounds and implicate f-actin proteins in this process. We, therefore, investigated changes in f-actin in SH-SY5Y human neuroblastoma cells exposed to 0.1 and 1 mM paraoxon, parathion, phenyl saligenin phosphate (PSP), tri-ortho-tolyl phosphate (TOTP), triphenyl phosphite (TPPi), and di-isopropyl phosphorofluoridate (DFP) for 0-48 h. The f-actin was measured by flow cytometry in cells labeled with Alexa 488 phalloidin. The relative amount off-actin was compared to total protein levels as determined by spectrophotometry. The cellular content of f-actin significantly decreasedfollowing exposure to PSP (0.1 mM, >30 min; 1 mM, >15 min), TOTP (0.1 mM, 16 h; 1 mM, >15 min), TPPi (1 mM, >4 h), paraoxon (1 mM, >24 h), and parathion (1 mM, 48 h). Exposure to DFP (0.1 and 1 mM) did not significantly alter f-actin content at any time point. Exposure to parathion (0.1 mM, 48 h) significantly increased the amount of cellular f-actin. Total protein was significantly decreased after exposure to PSP (0.1 and 1 mM, >8 h) and TPPi (1 mM, 48 h). Significant increases in total protein were observed following exposure to parathion (0.1 mM, >3 h). Consistent alterations in the protein content of DFP-exposed samples were not observed. These results suggest that the loss off-actin is an early event following OP compound exposure and that this loss significantly precedes a loss of protein content for some OP compounds (PSP, TPPi). Results also imply that under other exposure conditions (TOTP, paraoxon, parathion) alterations in the f-actin content are independent of protein content.