MOLECULAR NEUROTOXICOLOGY RESEARCH CORE

DESCRIPTION

The overall goal of this research core is to study the biochemical mechanisms of neurotoxicity and to monitor the neurotoxic effects of selected agrochemicals using molecular, cellular and developmental systems. The focus is on processes that affect cell death, gene expression, phosphorylation and excitability of the nervous system. The research projects use the tools of the Facility Cores to monitor the effects of pesticides and study mechanisms of their action with emphases on induced developmental abnormalities, myopathies, neuropathies such as Organophosphate Induced Delayed Neuropathy (OPIDN) and Alzheimer's Disease. Chemicals to be studied include organophosphates (OPs), carbamates, nicotinoids, pyrethroids and chlorinated insecticides.

CORE DIRECTOR AND MEMBERS

Isaac Pessah, Ph.D., Core Leader

Professor, Department of Molecular Biosciences

One Shields Avenue

University of California

Davis, CA 95616

bwwilson@ucdavis.edu

Fumio Matsumura, Ph.D.

Professor, Department of Environmental Toxicology

Barry Wilson, Ph.D.

Professor, Departments of Environmental Toxicology & Avian Sciences

P. Richard Vulliet, Ph.D., D.V.M.

Professor, Department of Molecular Biosciences

KEY WORDS

Neurotoxicology, organophosphates, cholinesterase, pesticides, development.

PROGRESS REPORT

INVESTIGATOR: Barry W. Wilson

Story of Discovery: This project, supported in part by the UCD NIOSH Agricultural Health and Safety Center, was chosen by NIH (NIEHS Letter, June 8, 1998) as a Story of Discovery to be presented to Congress to show how an important public health outcome was the result of many years of federal support. Selected were our studies showing that many clinical laboratories were using a commercial acetylcholinesterase (AChE) assay that underestimated enzyme activity by as much as 40 percent. The NIEHS noted our efforts with State regulatory agencies to bring practice in line with the public health intent of the law (see Wilson et al, 1997).

Cholinesterase (ChE) Methods: Fingersticks: We found that fingerstick assays and venous blood draws yielded statistically identical means when done under laboratory and clinic conditions Presumably pre-exposure fingerstick tests from more than 900 residents of migrant housing centers taken by our laboratory team also yielded statistically similar means. Later in the season some of the values taken by interviewers were lower than expected. These results are currently being analyzed for clues on how to train laypersons to sample blood accurately in the field. Spray Applicator Study: A study of spray applicators in Arkansas is underway with Dr. Howard Frumkin, Emory University and medical student Ashley Harmon. Pre-exposure venous blood draws from more than 50 spray applicators, shipped across the country and assayed in our laboratory had means that were the same as blood draws from our laboratory staff. Post-season samplings are in progress. The results to date indicate that variability in cholinesterase measurements can be much reduced if care is taken in the sampling, storage and assaying of the samples. Cal EPA ChE Guidelines: Cal EPA is finalizing their recommendations for cholinesterase measurements based on the venous blood draw method from our laboratory which is, in turn, based on the original method of Ellman and colleagues in 1961. Test-Mate Kit: We continue to examine the portable Test-Mate Kit. A new model purchased by the military for field use was according to our tests not accurate below 20 degrees C precluding its direct use in fields and farms under some weather conditions. However, it may be possible to place Test-Mate Kits in clinics where temperatures are within the accurate range of the instrument. Chemical Mixtures: A Gulf War study of possible interactions between antiChE organophosphates (sarin, paraoxon) and the carbamate protectant pyridostigmine is underway in collaboration with Dr. Peter Spencer, Oregon Health State University. Facilities have been approved, methodologies tested and the experiments are ready to go. The results will bear on both the events of the Gulf War and, in general, risks of those exposed to organophosphates including pesticides. Graduate student Ellen Coatney continued her study of the organophosphate DFP and cadmium using cultured muscle and nerve cells and embryos. Evidence for potentiation has been obtained with the cell cultures. Embryo work is in progress. A project has been initiated in collaboration with Chevron to study the combined effects of two triphenyl phosphite fuel additives examining the effects of oral dosing on chickens, using AChE, Neuropathy Target Esterase and neuromorphology. OPs , Neural and Muscle Damage: A collaborative study of the organophosphate diazinon and developing Medaka fish embryos was completed by Jon Hamm ( an aquatic toxicology student with Dr. David Hinton). Evidence that diazinon damages the developing neural retina led to the choice of Hamm's Ph.D. paper for a recent cover of Neurotoxicology. Finally, a project with University of Chicago scientists and Dr. Gerald Gronert (UCD Medical School) investigated the risks during surgery from muscle relaxants that are also cholinesterase inhibitors.

Neurotoxicity, Reproductive Toxicity and MTBE: Reports of reproductive toxicity from organophosphate pesticides were followed up by a pilot project with Center Investigator Lynn Wiley and her student Victoria Burrel obtaining evidence that methamidophos-treated sperm caused abnormalities in developing mouse embryos. We planned to use the fecal testosterone ELISA assay developed by Ph.D. student Joseph Billitti in collaboration with Center Investigator Bill Lasley to study whether OPs will affect reproductive state in mice. Instead, this year we participated in a crash UCD state funded special program to investigate risks from the fuel additive MTBE. We found no significant effects on fecal testosterone levels or testes morphology due to MTBE and its breakdown products at levels of MTBE as high as oral doses of 4000 mg/kg. Outreach: Wilson participated in a pilot outreach meeting in Salinas designed to Train the Trainers designed by Dr Pat O'Connor-Marer and his staff. He also gave talks on pesticide exposures and the fundamentals of toxicology in a workshop on toxicology for public health professionals sponsored by the Center for Occupational and Environmental Health in Parlier, CA. Word has spread about this project to standardize cholinesterase measurements. Wilson has been advising Dr. Spencer's staff on setting up blood cholinesterase measurements of Gulf War Veterans. Requests for advice have come from as far as the Lesser Antilles where Wilson is advising Dr Eddy Balenten as he sets up clinical measurements of blood cholinesterases.

Next Year: Studies on chemical mixtures, sarin and pyridostigmine, DFP and cadmium, work improving fingerstick measurements will continue. We look forward to the publication of the guidelines for cholinesterase testing by CA EPA . We anticipate that the next step will be a round robin validation study with volunteer research and clinical laboratories, followed by a request for the establishment of a clinical standard by the American College of Clinical Pathology.

PRESENTATIONS

Wilson BW, Lasley BL, Billitti JE and Faulkner BC 1998 MTBE and reproduction in male mice. 11th Annual Research Symposium, University of California Toxic Substances Research and Teaching Program. April 24-25, 1998 , Radisson Hotel Berkeley Marina, Berkeley CA

Billitti JE, Wilson BW, Lasley BL and Faulkner BC 1998 MTBE and reproduction in male mice. NorCal SETAC 8th Annual Meeting, June 22-23, 1998, Reno, Nevada

Wilson BW, Henderson JD, McCarthy SA, Billitti JE and McCurdy SA 1998 Fingerstick measurements of blood cholinesterases from California migrant housing center residents. SETAC 19th Annual Meeting, Charlotte NC, 15-19 November, 1998, PMP 040

Billitti JE, Faulkner BC, Lasley BL and Wilson BW 1988 Assessment of the potential male reproductive toxicity of the gasoline additive methyl tert-butyl ether utilizing a testosterone biomarker. SETAC 19th Annual Meeting, Charlotte NC, 15-19 November, 1998, PWA 212

Wilson BW, Henderson JD, McCarthy SA, Billitti JE and McCurdy SA 1998 Fingerstick measurements of blood cholinesterase from residents of California migrant housing centers. 4th International Symposium: Rural Health in a Changing World, Saskatoon Canada, October 18-22, 1998

Wilson BW, Henderson JD, McCarthy SA, Billitti JE and McCurdy SA 1998 Fingerstick based assays of cholinesterase levels. Conference on Federally Sponsored Gulf War Veteran's Illnesses Research, Pentagon City, VA, June 17-19, 1998.

Wilson BW and Spencer PS 1998. Low-level sarin neurotoxicity and its modulation by pyridostigmine. Bioscience Review, USAMRMD, May 31-June 4, Marriott's Hunt Valley Inn, Hunt Valley, Maryland

Wilson BW 1998 Standardizing cholinesterase measurements for humans, laboratory animals and wildlife. Invited Paper. ChE 1998 March 20-24, 1998 LaJolla CA.

Wilson BW 1998 Standardizing cholinesterase measurements for humans, laboratory animals and wildlife. Invited Presentation. ChE1998, LaJolla, CA March 20-24, 1998.

Wilson BW, Henderson JD, McCarthy SA, Billitti JE and McCurdy SA 1988 Fingerstick measurements of blood cholinesterase from residents of California migrant centers, 4th International Symposium: Rural Health and Safety in a Changing World, Saskatoon, Canada, October 18-22, 1988.

McCurdy SA, Beaumont JJ, Wilson BW, Henderson J, Samuels SJ, Morrin LA, and Carroll D. 1988. Work-related injury among California migrant Hispanic farm workers. , 4th International Symposium: Rural Health and Safety in a Changing World, Saskatoon, Canada, October 18-22, 1988.

PUBLICATIONS

Wilson BW, Sanborn JR, O'Malley MA, Henderson JD and Billitti RS 1997 Monitoring the pesticide-exposed worker. Occupational Medicine 12:347-363.

Billitti JE, Lasley BL and Wilson BW 1998 Development and validation of a fecal testosterone biomarker in mus musculus and peromyscus maniculatus. Biology of Reproduction 59:1023-1028.

Hamm JT, Wilson BW and Hinton DE 1998 Organophosphate-induced acetylcholinesterase inhibition and embryonic retinal necrosis in vivo in the teleost (Oryzias latipes) NeuroToxicology 19(6):853-870.

Wilson BW 1998 Ecosystem Health: Some Perspectives, in Multiple Stresses in Ecosystems, Cech, JJ Jr, Wilson BW and Crosby DG, Editors Lewis Publishers, Boca Raton, pp 53-57.

Seifert J and Wilson BW 1998 Phenyl valerate carboxylesterases of chick embryo brain as a marker for a new microassay of phospholipase A2. Analytical Letters 31(4):601-611.

Wilson BW 1998 Cholinesterase Inhibition in Encyclopedia of Toxicology, Vol 1, Wexler P, Editor in Chief, Academic Press, San Diego, pp 326-340.

Wilson BW, Sanborn JR, O'Malley MA, Henderson JD and Billitti JR 1997 Monitoring the pesticide-exposed worker. Occupational Medicine: State of the art Reviews, 12:347-361.

IN PRESS

Wilson BW, McCurdy SA, Henderson JD, McCarthy SA and Billitti JE. 1999 Cholinesterases and Agriculture: Humans, Laboratory Animals, Wildlife. In Cholinesterases 1998, Doctor BP, Quinn DM and Taylor P, Editors. Plenum Pub, NY

Wilson BW 1999 Cholinesterases in The Clinical Chemistry of Laboratory Animals, Loeb WF and Quimby FW, Editors, Pergamon Press, NY

Wilson BW, Henderson JD and Spencer PS 1999 Clinical effects of low-level exposures to chemical warfare agents in mice and chickens. Drug and Chemical Toxicology.

OUTREACH AND ADVISORY ACTIVITIES

Extending Pesticide Information and Resources to Health Care Providers. Salinas, November 12, 1998. Wilson presented information on Cholinesterases

Agricultural Pesticides and Health in California, October 15-16, Parlier CA Wilson presented introductory toxicology and wildlife exposure information

Wilson BW Subcommittee on Chronic Reference Doses for Selected Chemical Warfare Agents. National Research Council on Toxicology and Risk Assessment Program, Washington DC 1997-1998.

Wilson BW, Member California Pesticides Advisory Committee, Current

COLLABORATORS

ChE Projects:

Patrick J. O'Connor-Marer, UCD, Co-Investigator

Michael J O'Malley, UCD; Cal EPA; Co-Investigator

Jim Sanborn, Cal EPA, Co-investigator

John D. Henderson, (Wilson Lab) Research Associate

Joseph E. Billitti, (Wilson Lab) Post Graduate Researcher

Susan A. McCarthy, (Wilson Lab)Research Associate

Georgino Honorato deOliveira (Wilson Lab) Visiting Professor

Stephen McCurdy, UCD, Collaborator

Other Projects:

Gerald Gronert (Professor, UC Davis School of Medicine)

Howard Frumkin, Emory University Professor

Ashely Harmon , Emory University Medical Student

Peter Spencer, Director Neuroscience, Oregon State Health University

Eddy Balinten, Netherlands Lesser Antilles

INVESTIGATOR: Isaac N. Pessah

Title: Common Immunophilin Mechanism for Noncoplanar PCBs and Naturally Occurring Bromotyrosines from Ianthella basta

Significance: We recently provided evidence that noncoplanar PCBs with two (e.g., PCB 4) or three (e.g., PCB 95) ortho-chlorine substitutions possess nanomolar potency toward mobilizing Ca2+ from microsomes isolated from rat hippocampus, cerebral cortex, and cerebellum (1-3). A highly selective, structurally specific interaction between noncoplanar PCBs and RyR was found to fully account for altered microsomal Ca2+ transport. These actions of non-coplanar PCBs are mediated through interaction with FKBP12 since PCB-induced Ca2+ mobilization could be selectively eliminated by the immunosuppressant agents FK506 and rapamycin which affect dissociation of the FKBP12/RyR heterocomplex (3). An important observation is that unlike immunosuppressants FK506 and rapamycin, noncoplanar PCBs do not promote dissociation of the FKBP12/RyR complex, suggesting the existence of a novel modulatory site. We reported this year on the molecular mechanism by which noncoplanar PCBs alter Ca2+ regulation at the level of single channels and disrupt Ca2+ signaling in intact cells in a manner indistinguishable from those measured with naturally occurring bromotyrosine derivatives from the marine sponge Ianthella basta (4-6). These results suggest that PCB-related changes in neuroplasticity and spatial learning and memory we have shown to occur (7,8) could, at least in part, be mediated by alterations in FKBP12/RyR function. Common features inherent within the structure of noncoplanar PCBs and naturally occurring bastadins may underlie a common mechanism by which they alter Ca2+ dependent cell signaling by interacting with FKBP12.

References: Wong, P. W. and Pessah, I. N. Ortho-substituted polychlorinated biphenyls alter calcium regulation by a ryanodine receptor mediated mechanism. Structural specificity toward skeletal and cardiac type microsomal calcium release channels. Molec. Pharmacol. 49, 740-751, 1996.

Wong, P. W., Brackney, W. R., and Pessah, I. N. Ortho-substituted polychlorinated biphenyls (PCBs) alter microsomal calcium transport by direct interaction with ryanodine receptors of mammalian brain. J. Biol. Chem. 272, 15145-15153, 1997.

Wong, P. W. and Pessah, I. N. Non-coplanar PCB 95 alters microsomal Ca2+ transport by an immunophilin FKBP12-dependent mechanism. Molec. Pharmacol. 51, 693-702, 1997.

Pessah, I. N., Molinski, T. F., Meloy, T. D., Wong, P. G., Allen, P. D., Mohr, F.C. and Mack, M. M. Bastadins relate ryanodine-sensitive and -insensitive "leak" Ca2+ efflux pathways in skeletal muscle sarcoplasmic reticulum and BC3H1 cells. Amer. J. Physiol.: Cell Physiol. 272, C601-614, 1997.

Franklin, M. A., Penn, S. G., Lebrilla, C. B., Lam, T.H., Pessah, I. N., and Molinski, T. F. Bastadin 20 and Bastadin O-sulfate esters from Ianthella basta. Novel modulators of the Ry1R FKBP12 complex. J. Nat. Prod. Chem. 59, 1121-1127, 1997.

Wong, P. W., Joy, R. M., Albertson, T. E., Schantz, S. L., and Pessah, I. N. Ortho-substituted 2,2'3,5',6-pentachlorobiphenyl (PCB 95) alters rat hippocampal ryanodine receptors and neuroplasticity in vitro: Evidence for altered hippocampal function. NeuroToxicology 18, 443-456, 1997.

Schantz, S. L., Seo, B. W., Wong, P. W., and Pessah, I. N. Long-term effects of developmental exposure to 2,2'3,5',6-pentachlorobiphenyl (PCB 95) on locomotor activity, spatial learning and memory and brain ryanodine receptors. NeuroToxicology 18, 457-468, 1997.

Kiselyov, K., Xu, X., Mozhayeva, G., Kuo, T., Pessah, I.N., Mignery, G., Zhu, Z., Birnbaumer, L., and Muallem, S. (1998) Functional interaction between InsP3 receptors and store-operated Htrp3 channels. Nature 396, 478-482.

Narasimhan, K., Pessah, I. N., and Linden, D. J. (1998) Inositol-1,4,5-trisphosphate receptor-mediated Ca2+ mobilization is not required for the induction of cerebellar long-term depression in reduced preparations. J. Neurophysiol. 80, 2963-2974.

Title: Mechanisms of Delta-Hexachlorocyclohexane Toxicity: I. Relationship Between Altered Ventricular Myocyte Contractility and Ryanodine Receptor Function.

Significance: Hexachlorocyclohexanes (HCH) are commonly used as insecticides. However, these compounds have also been shown to be toxic to mammals. Previous studies have revealed that HCHs alter Ca2+ homeostasis in a variety of excitable and non-excitable cells and alter contractility of cardiac muscle. The stereoselective effects of the delta and gamma isomers of HCH were investigated on isolated ventricular myocytes from guinea pig and on single cardiac ryanodine receptor (RyR2) Ca2+ release channels from cardiac SR vesicles. Intracellular Ca2+ transients were examined in electrically stimulated cells using the fluorescent dye indo-1, and twitch contractions of myocytes were analyzed using a video-based edge motion detection system. Exposure of myocytes to 1-10 uM delta- but not gamma-HCH depressed the peak of intracellular Ca2+ transients and prolonged recovery time. These effects were correlated with the ability of delta-HCH to inhibit the binding of [3H]ryanodine, a conformationally sensitive probe for RyR2 function, to SR preparations (IC50 of 2 and 18 uM). Measurements of single channel gating kinetics under voltage clamp provide direct evidence of stereoselective enhancement channel open probability by delta-HCH. Results from these studies reveal that delta-HCH alters Ca2+ homeostasis and contractility in cardiac myocytes and the mechanism can be ascribed, at least in part, to a direct interaction with RyR2.

Analysis of channel gating kinetics in the presence of delta-HCH also revealed a non-fluctuating membrane current which remained even after RyR2 channels were blocked. We further elucidated the nature of a direct interaction between delta-HCH and biological membranes by measuring ionic currents across planar lipid bilayers made from defined lipids lacking cellular protein using voltage clamp. DMSO, in the presence or absence of 50uM gamma-HCH (lindane) or delta-HCH, produced negligible steady-state current with symmetric 100mM CsCl in the range of plus or minus 50mV. However, addition of 50uM Ca2+ to the bilayer chamber in the presence of delta-HCH induced a profound increase in ionic permeability which was not seen in the presence of gamma-HCH or DMSO control. Significantly, the permeability increase: (1) was proportional with increasing Ca2+ to ~ 600uM and saturated between 1 and 2mM Ca2+ regardless of holding potential, (2) occurred only when delta-HCH and Ca2+ were added to the same side of the membrane, and (3) was independent of the order of addition or to which side of the membrane delta-HCH and Ca2+ were added. The Ca2+-dependent current produced by delta-HCH was highly selective for monovalent cations (K+ >>Cs+ >Na+), with negligible conductance for Ca2+ or Cl-. In symmetric 100 mM K+, the conductance induced with 50 uM each of delta-HCH and Ca2+ was 4.25 pA/mV. The results show that delta-HCH increases the ionic permeability of phospholipid membranes by two distinct Ca2+-dependent mechanisms, one mediated through RyR, the other by a unique ionophore activity.

References: Buck, E. D., Lachnit, W. L., and Pessah, I. N. ( 1999) Delta-hexachlorocyclohexane induces Ca2+ release from cardiac sarcoplasmic reticulum by a ryanodine receptor mechanism insensitive to dantrolene. J. Pharmacol. Exp. Therap. (in press).

Buck, E. D., and Pessah, I. N. (1999) Delta-hexachlorocyclohexane mobilizes microsomal Ca2+ by ryanodine receptor dependent and independent mechanisms. J. Pharmacol. Exp. Therap. (in press).

INVESTIGATOR: Fumio Matsumura

Complementary DNA sequences of genes encoding calmodulin, partial structures of calmodulin-dependent protein kinase II (CaM-kinase II) and an L-type-like calcium channel al subunit (IVS5-IVS6-EF hand region) were identified and compared between susceptible and kdr strains of German cockroach, Blatella germanica. For this purpose, polymerase chain reactions (PCR) were used to obtain their sequences using cDNA from poly(A) + RNA isolated from their heads and thoraces. No mutation differences were found in all three sequences of calcium-regulating proteins between susceptible and strain. Northern blot analysis, however, showed reduced expressions of CaM-kinase II mRNA in two kdr strains. Western blot analysis with an antibody preparation against CaM-kinase II on protein levels confirmed above strain difference in the titer of this enzyme. In contrast, the levels of calmodulin as well as that of an L-type-like calcium channel gene expression were not different between susceptible and kdr strains.

Reference: Inagaki S, Koichiro K, Dunlap DY, and F Matsumura. 1998. Sequences of cDNAs encoding calmodulin, and partial structures of calmodulin kinase, and a calcium channel of kdr-resistant and ­susceptible German cockroaches, Blattella germanica. Comparative Biochemistry and Physiology Part C 120: 225-233.

INVESTIGATOR: P. Richard Vulliet

Staurosporine Induced Cytotoxicity

Recent work has shown that the protein kinase inhibitor staurosporine is able to facilitate neurite expression in pheochromocytoma (PC12) cells derived from the rat adrenal medulla. The neurite expression has been shown to be quite different than nerve growth factor (NGF) induced differentiation/neurite expression in PC12 cells. Firstly, staurosporine is able to induce neurite expression within several hours as opposed to NGF which requires days. The neurites are also colchicine resistant, neurotrophin receptor and protein kinase C independent, and has been suggested to be mediated via JNK kinase isoform.

Work in our lab has studied the kinetics of neurite expression using staurosporine. Interestingly, we have found the neurite expression to be maximal at 3-4 hours and increasing cytotoxicity was observed following this timepoint. At 24 hrs following addition of 100 ng/ml staurosporine it was observed that most cells had experienced toxicity and loss of neurites. Furthermore, it was observed that cell death was exacerbated in Krebs-Ringer-Hepes buffer containing high calcium (2.6mM). This suggests that staurosporine treatment may initiate/facilitate calcium-induced exitotoxicity in conjunction with staurosporine-specific toxicity. Or, it suggests that calcium-induced toxicity and staurosporine-induced toxicity utilize the same signal transduction pathway within PC12 cells.