Charaterization of Chlorpyrifos Toxicity on the Pancreatic Beta Cell Line Rinm5f

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Charaterization of Chlorpyrifos Toxicity on the Pancreatic Beta Cell Line RINm5f

Zhongyu Yan Wright State University

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CHARACTERIZATION OF CHLORPYRIFOS TOXICITY ON THE

PANCREATIC BETA CELL LINE RINM5F

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy

ZHONGYU YAN M.D., Tianjin Medical School, 1996 M.S., Capital University of Medical Sciences, 2003

2010 Wright State University WRIGHT STATE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

I HEREBY RECOMMEND THAT THE DISSERTATION PREPARED UNDER MY SUPERVISION BY Zhongyu Yan ENTITLED Characterization of Chlorpyrifos Toxicity On The Pancreatic Beta Cell Line RINm5f BE ACCEPTED IN PARTIAL FUFILLMENT OF THE REQUIRMENTS FOR THE DEGREE OF Doctor of Philosophy .

David R. Cool, Ph.D. Dissertation Director

Gerald M. Alter, Ph.D. Director, Biomedical Sciences Ph.D. Program

Andrew T. Hsu, Ph.D. Dean, School of Graduate Studies

Committee on Final Examination

David R. Cool, Ph.D.

Thomas L. Brown Ph.D.

James E. Olson Ph.D.

Daniel T. Organisciak Ph.D.

Courtney Sulentic Ph.D.

ABSTRACT

Yan, Zhongyu. Ph.D., Biomedical Sciences Ph.D. Program, Wright State University, 2010 Characterization of Chlorpyrifos Toxicity on the Pancreatic Beta cell line RINm5f

Chlorpyrifos (CPF) is a wellknown organophosphate (OP) insecticide that inhibits acetylcholinesterase (AChE). Organophosphates have been shown to induce hyperglycemia in both humans and animals that cannot be explained by AChE inhibition alone. Pancreatic βcells are responsible for secreting insulin and playing a fundamental role in glucose homeostasis. The aim of this research was to investigate

CPF toxicity on the insulin secreting βcell line RINm5f (RIN). In the first part of this dissertation, we determined that cytotoxic concentrations of CPF between 50 µM and

200 µM induced a dosedependent increase in RIN cell apoptotic and necrotic death

(p<0.05). Lower doses, i.e., 525 µM did not induce apoptosis or necrosis. In further mechanistic studies, inhibitors of cJun Nterminal kinase (JNK) and P38 pathways, i.e.,

SP600125 and SB202190, successfully attenuated CPFinduced apoptosis (p<0.05), suggesting cell death is mediated by JNK and/or p38. In addition, NAcetylLcysteine

(NAC), an antioxidant, inhibited apoptosis suggesting that oxidative stress is also involved in RIN cell death. In the second part of this dissertation, low dose CPF’s effects on insulin synthesis and secretion was studied. Our results showed that RIN cells treated with CPF for 4 hr exhibited a dosedependent decrease in insulin secretion in response to stimulation by 50 mM potassium (p<0.05). Insulin secretion stimulated by iii

25 mM glucose also showed significant decreases upon incubation with various doses of CPF (p<0.05). Insulin synthesis involves proteolytic cleavage of proinsulin at two specific sites. The enzymes responsible for promoting proinsulin cleavage, i.e, prohormone convertase (PC) 1/3 and 2 (PC2), contain a serine hydrolase catalytic triad,

HisAspSer, similar to AChE. CPF modification of PC enzymes similar to AChE, could potentially interrupt the conversion of proinsulin to insulin and disrupt insulin secretion. PC2 enzyme activity was significantly inhibited by CPF at concentrations as low as 10 µM (p<0.01). RIN cells, for the first time, were found to secrete proinsulin was through a constitutive instead of a regulated pathway. In summary, our results show that CPF not only exerts a direct cytotoxic effect at concentrations above 50 µM, but also significantly impairs insulin secretion at lower doses. Low dose CPF could potentially affect endocrine function in vivo by inhibiting the proinsulin processing enzyme PC2.

TABLE OF CONTENTS

Page

I. INTRODUCTION…………………………………………………….. 1 Chemical and Physical Properties of Chlopyrifos…………………. 1 Use of CPF………………………………………………………... 3 Biotransformation of CPF ……………………………………….. 3 Exposure to CPF………………………………………………….. 5 CPF Toxicity……………………………………………………… 5 CPF and Hyperglycemia………………………………………….. 10 CPF and Pancreas Toxicity………………………………………. 12 CPF and Insulin Secretion………………………………………... 13 Hypothesis and Specific Aims…………………………………… 22 II. MATERIALS AND METHODS……………………………………. 23 CPF Preparation………………………………………………….. 23 Cell Culture Medium…………………………………………….. 23 Cell Culture……………………………………………………….. 23 MTT Assay………………………………………………………... 23 Cell Apoptosis Assay……………………………………………... 24 Cell Necrosis Assay………………………………………………. 27 Insulin Secretion…………………………………………………... 29 Insulin Detection by ELISA Assay……………………………… 29 Proinsulin Detection by ELISA Assay…………………………... 30 Western Blotting Analysis for Prohormone Convertase 1 and 2 30 Detection of Prohormone Convertase 2 Activity………………... 32 PC2 Activity Detection by MALDITOF MS Analysis………… 33 Inhibitor Study for Apoptosis and Necrosis……………………... 34 III. RESULTS……………………………………………………………… 36 Cytotoxicity detection by MTT………………………………….. 36 Cell Apoptosis Assay…………………………………………….. 38 Cell Necrosis Assay……………………………………………… 40 The Percentage of Apoptosis and Necrosis of RIN Cells Treated with CPF………………………………………………………….. 43 Antioxidant NAcetylLCysteine on Cell Viability…………….. 44 Inhibition of p38 and JNK MAPK on Cell Viability…………... 47 Basal Insulin and Proinsulin……………………………………... 52 Stimulated Insulin and Proinsulin Secretion by Potassium…….. 55

Stimulated Insulin and Proinsulin Secretion by Glucose……….. 60 Intracellular Insulin and Proinsulin after Potassium Stimulation. 63 Intracellular Insulin and Proinsulin after Glucose Stimulation… 66 PC Enzyme Expression in RIN Cells…………………………… 68 PC2 Enzyme Activity…………………………………………….. 71 IV. DISCUSSION…………………………………………………………. 75 ChlorpyrifosInduced Cell Death……………………………….. 75 Oxidative Stress induction by CPF……………………………... 76 Involvement of the JNK and p38 Pathway in CPFInduced Cell Death……………………………………………………….. 77 Effect of CPF on Insulin Processing and Secretion…………… 80 V. CONCLUSIONS……………………………………………………… 87 VI. FUTURE STUDY…………………………………………………….. 88 Molecular Targets of JNK and p38……………………………. 88 PC1 Activity Detection…………………………………………. 88 Prohormone Convertase Catalytic Serine Phosphorylation by CPF……………………………………………………………… 88 Insulin Secretion Changes in Primary βcell line and In Vivo Study…………………………………………………………….. 89 VII. REFERENCES………………………………………………………... 90

LIST OF FIGURES

Figure Page 1. Chemical Structure of CPF…………………………………………... 1 2. Schematic Drawing of OP on the Inhibition of Acetylcholine Signaling……………………………………………………………... 2 3. Biotransformation of CPF…………………………………………… 4 4. Known Primary and Secondary Targets of Organophosphates…….. 11 5. Endocrine Pancreas…………………………………………………. 14 6. Primary Structure of Human Preproinsulin…………………………. 17 7. Cleavage of Proinsulin by PC enzymes……………………………… 18 8. Structure Comparison of Acetylcholinesterase and PC enzymes……. 20 9. Components of Cell Death Detection ELISA………………………... 25 10. CPF Decreases MTT metabolism in RIN Cells……………………… 37 11. CPF Induces RIN Cell Apoptosis in RIN Cells……………………… 39 12. CPF Induces Necrotic Cell Death in RIN Cells……………………… 41 13. The Effects of Antioxidant NAcetylLcysteine (NAC) on CPFInduced RIN Cell apoptosis……………………………………. 45 14. The effects of Antioxidant NAcetylLcysteine (NAC) on CPFInduced RIN Cell Necrosis……………………………………... 46 15. The p38 Inhibitor SB202190 Inhibits CPFinduced RIN Cell Apoptosis…………………………………………………………….. 48 16. The Effects of p38 Inhibitor SB202190 on CPFInduced RIN Cell Necrosis……………………………………………………………… 49 17. The JNK Inhibitor SP600125 Inhibits CPFInduced RIN Cell Apoptosis…………………………………………………………….. 50 18. The JNK inhibitor SP600125 Inhibits CPFinduced RIN Cell Necrosis……………………………………………………………… 51 19. Effect of CPF on Basal Insulin Secretion……………………………. 53

vii

20. Effect of CPF on Basal Proinsulin Secretion…………………………. 54 21. Effect of CPF on PotassiumStimulated Insulin Secretion…………… 56 22. Effect of PotassiumStimulated Proinsulin Secretion………………... 58 23. Effect of GlucoseStimulated Insulin and Proinsulin Secretion……. 61 24. Changes of Intracellular Insulin after 50 mM Potassium Stimulation... 64 25. Changes of Intracellular Proinsulin after 50 mM Potassium Stimulation…………………………………………………………... 65 26. Changes of Intracellular Insulin and Proinsulin after Glucose Stimulation…………………………………………………………. 67 27. PC1 and PC2 Expression in RIN Cells……………………………….. 69 28. PC2 Enzyme Activity Determination after 4 hr Exposure to CPF……. 72 29. Mass spectrometry analysis on PC2 enzyme activity and its inhibitor.. 74 30. Scheme of Steps Involved in Potassium and Glucose Stimulated Insulin Secretion……………………………………………………... 82 31. Scheme of Proinsulin Processing in Normal β Cells Via Regulated and RIN Cells via Constitutive Secretory Pathway…………………. 84

viii

LIST OF TABLES

Table Page

1. Summary of In vitro Cell Viability Affected by CPF………………. 8 2. LDH Experimental Design………………………………………… 28

3. Antioxidant NAC Experimental Design…………………………… 34

4. P38 Inhibitor Experimental Design………………………………... 35 5. JNK Inhibitor Experimental Design……………………………….. 35

6. Summary of Percent Cell Death Affected by CPF…………………. 43 7. Insulin Stimulation by Potassium………………………………….. 57 8. Proinsulin Stimulation by Potassium………………………………. 59

9. Insulin Stimulation by Glucose……………………………………. 62

10. Proinsulin Stimulation by Glucose………………………………… 62

ACKNOWLEDGEMENTS

First of all, I would like to give the greatest thanks to my advisor Dr. David Cool.

Thank him for giving me the opportunity working in the lab, mentoring me through the Ph.D. journey. Without his scientific expertise and encouragement; this thesis could not be possible.

I am also grateful to my thesis committee, Dr. James Olson, Dr. Daniel

Organisciak, Dr. Thomas Brown and Dr. Courtney Sulentic for their valuable time and insightful comments. Their suggestions and criticism help my dissertation so much.

My thanks also go to Bev Grunden for her advice and help on my statistics analysis.

Very special thanks to Andrea Hoffmann for her contribution and support. She is always there for me not only academically but also in my personal life. My sincere thanks go to William Grunwald Jr., Sean Harshman, Curt Grigsby and Molly Davidson for all their assistance and friendship.

I also would like to thank many people from Pharmacology and Toxicology

Department at Wright State University including Dr. Mariana Morris, Dr. Nadja Grobe,

Catherine Winslow, Mary Key, Terry Oroszi, Laurie Schoettinger and Nathan Weir.

Thank you all for your support.

Many thanks go to Biomedical Sciences Ph.D. Program especially Dr. Gerald

Alter, Diane Ponder and Karen Luchin.

I would like to show my gratitude to my friends Fine Wu, Yili Wei, XiuFen and

Zemin Yu for their help and support for my family.

I would like to give my deepest appreciation to my family for all their love and

x encouragement. For my parents who support me in all my life. Most of all for my loving, supportive, encouraging, and patient husband, Rendong and my daughter,

Kaitlyn, this thesis is simply impossible without your loving support.

Lastly, I offer my regards and blessings to all of those who supported me in any respect during the completion of the thesis.

Background and Significance

Chemical and Physical Properties of Chlorpyrifos

FIG. 1. Chemical Structure of CPF

Chlorpyrifos (O, Odiethyl O3, 5, 6 trichlopyridin2pyridyl phosphorothioate)

(CPF) is one of the organophosphorous (OP) pesticides (Figure 1). It is well known that toxicity of OP pesticides is via persistent inhibition of an enzyme, acetylcholinesterase (AChE), in the brain and peripheral nervous system (Rosenfeld et al. 2006; Shenouda et al. 2009). AChE is primarily located in the postsynaptic membrane of central and peripheral cholinergic systems. The inhibition of AChE results in decreased degradation of a neurotransmitter, acetylcholine, leading to overstimulation of the associated synaptic systems (Figure 2). Symptoms of exposure include nausea, headaches, twitching, trembling, excessive salivation and tearing, inability to breathe because of paralysis of the diaphragm, convulsions, and at higher doses, death (Simpson et al. 2002).

FIG. 2. Schematic drawing of OP on the inhibition of acetylcholine signaling.

Uses of CPF

About 70% of insecticides used in the United States are OPs. Since first introduced into the marketplace in 1965, CPF has been widely used worldwide as an insecticide to control pests in agriculture, home, lawns and golf courses and to control mosquitoes

(EPA 2009). Residential use of CPF was eliminated in the United States in 2001(EPA

2001). However, CPF continues to be used to control crop damage from insects in agriculture. In 20022006, the United States accounted for 16% of total worldwide agriculture use of CPF and 26% with regard to nonagricultural use from just one company, i.e., Dow AgroSciences (Eaton et al. 2008). In California, 2.3 million pounds of CPF were reported sold in 2004 (California Department of Pesticide Regulation

2004)

Biotransformation of CPF

CPF undergoes biotransformation by different pathways and tissues in the body (Figure 3). Cytochrome P450 (CYP) is responsible for oxidative desulfuration of CPF to form chlorpyrifosoxon (CPO). CPO is responsible for inhibition of AChE.

The rate of desulfuration in the liver microsomes is 1000 times the rate in brain microsomal and crude mitochondrial fractions, i.e. 327 pmol/g/min vs. 428 nmol/g/min respectively (Chambers et al. 1989). CPO can be detoxified by oxonases, such as paraoxonase (PON) to form diethylphosphate (DEP) and

3,5,6trichloro2pyridinol (TCPy). CYP enzymes also catalyze dearylation of CPF to diethyl thiophosphate (DETP) and TCPy (Jokanovic 2001). Different isoforms of CYP enzymes have different capacities to convert CPF to its oxon form and to detoxify CPF.

FIG. 3. Biotransformation of CPF

CPF is activated to CPF oxon (CPO) by CYP (to the left) and then is detoxified by paraoxonase (PON). CYP enzymes catalyze dearylation of CPF to the inactive metabolites i.e., DEPT, DEP and TCPy.

TCPy and glucoronide and sulfate conjugates of TCPy are inactive metabolites of

CPF and are excreted in the urine and feces.

Exposure to CPF

Exposure to CPF can be through direct dermal contact, contact with treated surfaces, ingestion of CPF contaminated dust, and by breathing air inside or outside treated buildings (Pesticide Action Network North America 2006). Exposure also results from eating food contaminated with CPF residues (Fenske et al. 2002). Data from the Center for Disease Control and Prevention (CDC) showed that 93% of U.S. residents sampled between 1999 and 2000 had CPF in their bodies. CPF is found in higher levels (nearly 2 x) in children compared with adults (Schafer et al. 2004). The reasons for this are believed to be that children have different activity patterns. For example children are closer to the ground and put more toys in their mouths (Bearer

1995; Landrigan et al. 1999; O'Rourke et al. 2000). They also consume more food and water per body weight than adults (Bearer 1995; Landrigan et al. 1999). In addition, children have a greater surface to volume ratio which can increase the absorption of

CPF or its metabolites from their skin (Bearer 1995; Landrigan et al. 1999; Needham et al. 2000).

CPF Toxicity

A wide variety of effects have been seen with CPF and CPO in in vitro studies.

These effects fall into 8 categories: cytotoxicity, effects on macromolecule synthesis

(DNA, RNA, proteins), interactions with neurotransmitter receptors, interactions with signal transduction pathways, effects on neuronal differentiation, interactions with

5 enzymes, neurotransmitter release or uptake, and oxidative stress (Eaton et al. 2008).

Micromolar concentrations of CPF were found to cause cytotoxicity in many in vitro studies (Table 1). In the human monocyte cell line, U937, CPF induced apoptosis in a time and dosedependent manner (Nakadai et al. 2006). Human placental choriocarcinoma (JAR) cells exhibited a dosedependent reduction in viability starting at a concentration of 31.25 µM (Saulsbury et al. 2008). Primary cortical neurons cultured from embryonic and newborn rats also showed apoptosis following exposure to CPF (Caughlan et al. 2004). However, no studies have been done to evaluate CPF cytotoxicity on pancreatic cells in vitro .

CPF and CPO were observed to inhibit macromolecule synthesis, in particular,

DNA synthesis at micromolar concentrations (Garcia et al. 2001; Qiao et al. 2001;

Guizzetti et al. 2005; Jameson et al. 2006). Garcia et al found that CPF and CPO induced a concentrationdependent inhibition in [ 3H] thymidine incorporation in astroglial cells (Guizzetti et al. 2005). Meanwhile, inhibition of DNA synthesis in an adrenal gland cell line, PC12, and glial cell line, C6, was also reported (Qiao et al.

2001). Although further studies are needed to investigate potential cellular and molecular mechanisms involved in the DNA inhibition, there is evidence that transcriptional events that mediate cell division and differentiation are interrupted. This includes decreased DNA binding activity of transcription factor AP1 in the presence of

CPF and CPO (Crumpton et al. 2000; Garcia et al. 2001). In regard to signal transduction pathways, CPF and CPO were found to inhibit adenylate cyclase, possibly through activation of muscarinic M2 receptors (Song et al. 1997; Aldridge, Justin E. et

6 al. 2003; Meyer et al. 2004; Meyer et al. 2005).

Evidence for interaction of CPF and CPO with cholinergic muscarinic receptors has been found (Liu et al. 1999; Tang et al. 1999; Qiao, D. et al. 2002; Richardson et al.

2004). Decreased density of muscarinic M2 receptors has also been observed (Liu et al.

1999; Tang et al. 1999; Qiao, D et al. 2002; Rhodes et al. 2004; Richardson et al. 2004).

In binding studies, CPF and CPO have been shown to displace agonists from muscarinic receptors (Huff et al. 1994). This suggests CPF and/ or CPO may interact directly with the receptors and downregulate them.

Many papers have demonstrated that CPF induces oxidative stress (Bagchi et al.

1995; Crumpton et al. 2000; Giordano et al. 2007; Buyukokuroglu et al. 2008).

Oxidative stress refers to an imbalance between the elevated levels of reactive oxygen species (ROS) and/or compromised function of the antioxidant defense system (Sies

. . 1997). ROS includes superoxide anion (O 2 ), hydroxyl radicals ( OH), and hydrogen peroxide (H 2O2). CPF has the ability to promote glutathione depletion, active oxygen formation, and lipid peroxidation in various in vitro preparations and in vivo studies.

Meanwhile, the addition of an antioxidant, prevented the production of lipid peroxidation in the presence of CPO (Qiao et al. 2005). Melatonin, an antioxidant that protects lipids, proteins and DNA from oxidative damage, suppressed CPFcaused

HepG2 (a human hepatocellular carcinoma cell line) cytotoxicity (Gultekin et al.

2006).

Table 1

Summary of In vitro cell viability affected by CPF

Cell line/ tissue Effects CPF concentration Reference

U937 cells Apoptosis 20 µM (Nakadai et al. 2006) (human mococyte cell line)

Rat cortical neurons Apoptosis 30 µM (Caughlan et al. 2004)

Human astrocytes Apoptosis 100 µM (Mense et al. 2006)

Human placental Apoptosis 60 µM (Saulsbury et al. 2008) choriocarcinoma

Human astrocytes Necrosis 0.2 µM (Mense et al. 2006)

U937 cells LDH release 70 µM (Nakadai et al. 2006) (human mococyte cell line)

Rat cortical neurons MTT reduction 15 µM (Caughlan et al. 2004)

Mouse CGNs MTT reduction 12 µM (Giordano et al. 2007) (cerebellar granule neurons)

Human placental MTT reduction 31.25 µM (Saulsbury et al. 2008) choriocarcinoma

1321N1 cells Trypan blue 50 µM (Guizzetti et al. 2005) (human astrocytoma cell line) exclusion

Hep G2 cells Decreased 500 µM (Cool et al. (human hepatoma cell line) proliferation upublished)

Pituitary rat GH3 cells Decreased 50 µM (Ghisari et al. 2005) proliferation

Hep G2 cells Increased 1 µM (Cool et al. (human hepatoma cell line) proliferation unpublished)

Pituitary rat GH3 cells Increased 10 µM (Ghisari et al. 2005) proliferation

In vivo toxicity of CPF includes neurotoxicity, carcinogenesis and reproductive toxicity. Many studies demonstrate that exposure to CPF interferes with development of mammalian nervous system (Aldridge et al. 2003; Aldridge et al. 2005; Slotkin et al.

2005; Slotkin et al. 2006). It is believed that oxidative stress is one of the mechanisms involved in OPinduced neurotoxicity (Slotkin et al. 2005), however, there are other mechanisms. For example, nontoxic exposure to CPF on gestational days (GD) 1720 or postnatal days (PN) 14 in rats evoked increases in serotonin (5HT) turnover which is associated with behavioral anomalies (Aldridge et al. 2005). A new analysis links children's attentiondeficit disorder with exposure to common pesticides including CPF.

The study included 1139 children from 8 to 15 years of age with average exposure to

OP pesticides. Children with higher levels of pesticide residue in their bodies had increased chances of having ADHD, attentiondeficit hyperactivity disorder, a common problem that causes students to have trouble in school (Bouchard et al. 2010).

Although no evidence of CPF carcinogenicity has been found by the U.S EPA, several human epidemiology studies have suggested possible links between CPF and lung and rectal cancers (Lee et al. 2004; Lee et al. 2007).

Reproductive toxicity also has received much attention. Farag et al. reported that male mice treated with 25 mg/kg CPF for 4 weeks before mating with untreated females was associated with decreased number of live fetuses, and increased number of dead fetuses (Farag et al. 2010). Exposure to CPF in utero has been shown to result in low birth weights and reduced head circumference of newborns especially for individuals who have low levels of PON1 (Whyatt et al. 2001; Berkowitz et al. 2004;

Whyatt et al. 2004; Furlong et al. 2006).

To date, the majority of CPF research has focused on the formation and metabolism of CPO, as this metabolite causes the irreversible inhibition of AChE activity through binding to serine at the enzyme active site. It should be recognized that there are many other serine hydrolases that are potential secondary targets for the oxon (Figure 4).

These enzymes are fatty acid amide hydrolase (FAAH), diacylglycerol (DAG) lipase, acylpeptide hydrolase (APH), NTElysophopholipase (NTElysoPLA), monoacylglycerol (MAG) lipase, and serine hydorlase KIAA1363 (Casida et al. 2004;

Casida et al. 2005; Nomura et al. 2006; Nomura et al. 2008). Several serine hydrolases were found to be inhibited by CPO at medium to high nanomolar concentrations, for example, serine hydrolase KIAA1363 at 8 nM, FAAH at 40 nM, APH at 82 nM and

NTElysoPLA at 180 nM. However, knowledge of the role of the majority of serine hydrolases and the consequences of their inhibition is still very limited.

CPF and hyperglycemia

Hyperglycemia is one of the effects of OPs in acute and subchronic exposures

(Gupta 1974; Zadik et al. 1983; Shobha et al. 2000; Abdollahi et al. 2004; Pourkhalili et al. 2009). In humans, OPrelated poisonings raised blood glucose up to five fold

(Namba et al. 1971; Hayes et al. 1978; Meller et al. 1981; Matin et al. 1982).

OPcaused hyperglycemia has been confirmed in laboratory animals as well (Dybing et al. 1958; Matin et al. 1982; Fletcher et al. 1988; Abdollahi et al. 2004; Pourkhalili et al.

2009). Although hyperglycemia by OPs is well demonstrated, the underlying mechanism is unknown. Previous research has mainly linked hyperglycemia to OP

FIG. 4. Known primary and secondary targets of organophosphates.

11 induced liver deregulation. Goel et al. indicated that CPF treatment induces a variety of alterations in carbohydrate metabolizing enzymes in rat tissues (Goel et al. 2006).

Specifically, they found a significant elevation of glycogen phosphorylase, decreased glycogen levels, significantly inhibited hexokinase activity which suppressed glycolysis, increased activity of glucose6phosphatase (increased rate of glycogenolysis) and decreased 14 Cglucose uptake. Rezg et al. showed that subchronic exposure to malathion (one of the OPs) significantly decreased hepatic protein and lipid contents which might be associated with increased liver gluconeogenesis (Rezg et al.

2007).

CPF and Pancreas Toxicity

In our laboratory, we are interested in the toxicity associated with the endocrine system, especially the pancreas. The pancreas is comprised of two secretory systems, exocrine and endocrine, that regulate two major physiological processes: digestion and glucose metabolism. The endocrine pancreas consists of four specialized cell types that are organized into compact islets embedded within acinar tissue (Figure 5). These cells secrete hormones into the bloodstream. PP and delta cells produce pancreatic polypeptide (PP) and somatostatin, respectively. The alpha and beta cells regulate the usage of glucose through the production of glucagon and insulin, respectively

(Bardeesy et al. 2002).

As important as the liver, the pancreas plays a critical role in regulating glucose homeostasis (Felig et al. 1976; Rasmussen et al. 1990; Schinner et al. 2005). In vivo studies performed in our laboratory found cutaneous CPF exposure caused rat

12 pancreatic tissue damage (Rutherford and Cool, unpublished data). Elevated levels of monocyte and leukocyte infiltration into pancreas suggested inflammation. Increased inflammatory mediators such as TNFα and IL1 further support this conclusion.

Although pancreatic βcells are responsible for secreting insulin in response to glucose, many studies on OPinduced pancreas toxicity have only focused on acinar cell injury related pancreatitis (Dressel et al. 1979; Moore et al. 1981; Dagli et al. 1983; Frick et al. 1987; Goodale et al. 1993; Gulalp et al. 2007). So far no studies have investigated the direct effect of CPF on pancreatic βcell viability.

CPF and Insulin Secretion

Organophosphate pesticides have been investigated for their interruption of insulin secretion. The pesticide, diazinon, was found to inhibit glucosestimulated insulin secretion from the isolated islets in rats (Pourkhalili et al. 2009). Another study from

Abdollahi et al. found that handpicked islets treated with 200 and 400 ppm malathion for 4 weeks showed decreased glucosestimulated insulin secretion up to 69.6%

(Abdollahi et al. 2004). More importantly, data from our laboratory found that there was a significant decrease in pancreatic insulin content and hyperglycemia in

CPFtreated rats, which couples CPF toxicity with hyperglycemia and possibly diabetes (Rutherford and Cool, unpublished data).

Insulin secretion by pancreatic βcells is a highlyregulated process, from transcription to translation, peptide modification and finally secretion. Any deviation from this pathway can result in conditions that lead to insulin secretion disorders.

FIG. 5. Endocrine pancreas

This drawing shows the relative size and distribution of cells in a normal pancreas.

Insulin is a heterodimeric protein with a 21 amino acid Achain and a 30 amino acid

Bchain linked by two disulfide bonds (Ryle et al. 1955). The insulin gene however encodes a larger molecule, preproinsulin (Sures et al. 1980; Ullrich et al. 1980) (Figure

6).

From preproinsulin to insulin, posttranslational processing is necessary in order to achieve maturation. Preproinsulin contains a signal sequence at its Nterminus, an Achain, Bchain C peptide (Ryle et al. 1955). The A and Bchains are linked by disulfide bonds. The signal peptide is to direct the protein to the lumen of the endoplasma reticulum and then is cleaved by signal peptidase to form proinsulin (Dev et al. 1990; Davidson 2004). During maturation, a critical step is the cleavage of proinsulin at pairedbasic amino acid residues by specific proteinases (Docherty et al.

1982). The prohomone convertase 1/3 (PC1/3) and 2 (PC2) are identified as the enzymes responsible for cleavage of proinsulin at pairedbasic amino acid sites, while

PC1 cleaves at the BC junction and PC2 cleaves at the AC junction (Figure 6 and 7)

(Roebroek et al. 1986; Mizuno et al. 1988; Fuller et al. 1989; Seidah et al. 1990;

Smeekens et al. 1990; Nakayama et al. 1991).

Conversion of proinsulin to insulin occurs mainly in the Golgi and insulin secretory granules (Steiner et al. 1967; Sorenson et al. 1970; Kemmler et al. 1973; Orci et al. 1973; Hutton et al. 1982; Docherty et al. 1983). Secretory granules have an acidic internal pH (between 5.0 and 6.0) and a high concentration of free calcium (1 to 10 mM)

(Hutton 1984). PC1 is synthesized as a precursor, preproPC1. There are 5 domains within the precursor: an Nterminal signal peptide; a prosequence flanked by basic

15 residues; a catalytic domain having the typical serine protease catalytic triad; a Homo B domain for folding and a Cterminal helix domain (Muller et al. 1999) (Figure 8).

The 87 KDa proPC1to the 84 KDa form occurs in the endoplasmic reticulum by autocatalysis (Goodman et al. 1994; Zhou et al. 1994). After delivery to secretory granules, further cleavage in the Cterminal region results in the mature 66 KDa form

(Zhou et al. 1994). It is enzymatically more active than the 84 KDa form. Disruption of the PC1 gene could cause multiple neuroendocrine and endocrine precursorprocessing defects (Zhu et al. 2002).

PC2 is also synthesized as a precursor. In contrast to PC1, PC2 prosequence cleavage occurs in the late Golgi and secretory granules instead of in the endoplasmic reticulum (Guest et al. 1992; Zhou et al. 1994). Newly synthesized PC2 folds quite slowly, taking approximately 1 h to exit from endoplasmic reticulum (Guest et al. 1992;

Creemers et al. 1996). The 76 KDa proPC2 conversion to the 66 KDa active enzyme is also autocatalytic and the activation requires an acidic pH (Lamango et al. 1999). One unique feature of proPC2 is that a specific chaperone, 7B2 is required to facilitate proPC2 maturation (Braks et al. 1994; Zhu et al. 1995). As PC1/3 and PC2 are responsible for the cleavage of proinsulin at paired basic amino acids (Figure 7), modifications that can cause alterations on PC enzyme activities will disrupt insulin maturation and further secretion.

The primary target of OPs is inhibition of AChE via phosphorylation of the catalytic triad in the AChE enzyme molecule (Figure 8). There are many secondary targets affected by OPs (Figure 4).

FIG. 6. Primary structure of human preproinsulin

Preproinsulin comprises a signal peptide ( clear ovals), Bchain ( orange ovals), connecting peptide (pu rple ovals), and Achain (blue ovals). The pair of basic residues is indicated (green ovals). The arrows repres ent the processing sites by PC1 /3 and PC2.

C Chain

A Chain PC1 Cleavage site

PC2 Cleavage site

B Chain

FIG. 7. Cleavage of proinsulin by PC enzymes

PC1 cleaves at BC junction and PC2 cleaves at AC junction of proinsulin to form insulin and C peptide.

OP’s effects are elicited through the phosphorylation of serine at the active site. AChE is a serine hydrolase enzyme similar to other subtilisin enzymes (Casida et al. 2004).

PC1/3 and PC2, that are responsible for cleaving proinsulin to insulin, are also serine hydrolases belonging to the subtilisin family (Seidah et al. 1999). As such, these enzymes also have a catalytic triad similar to AChE, i.e., Asp, His, Ser (Figure 8) with serine located at the active site. This triad is closely related to that of, AChE, i.e., Glu,

His, Ser. This relationship leads to the hypothesis that organophosphate compounds will inhibit the PC enzymes in a fashion similar to acetylcholinesterase. If OPs were to affect the catalytic triad in the proinsulin processing enzymes similar to how it affects

AChE, then they could have an effect on the processing of proinsulin to insulin, i.e., insulin maturation and secretion. However, there is no information available regarding the effects of CPF on insulin secretion. If insulin secretion is affected by CPF, is the impairment because of the inhibition of prohormone convertase by CPF? Either an alteration in the viability of pancreatic βcells, or disruption of insulin secretion by CPF, would have deleterious effects on insulinglucose regulation, providing an additional reason for OPcaused hyperglycemia.

Diabetes mellitus is a major health problem characterized by hyperglycemia resulting from defects in insulin secretion and/or action, currently affecting over 170 million people worldwide and prospectively affecting over 365 million by 2030

(Rathmann et al. 2004). Environmental factors are known to account for the development of diabetes (Yoon et al. 1987; Ferner 1992). With OPs adverse effects on

FIG. 8. Structure comparison of acetylcholinesterase and PC enzymes.

CPO phosphorylates AChE enzyme molecule at the catalytic triad GluHisSer, whereas PC enzymes also have a catalytic triad similar to AChE, i.e., AspHisSer with serine located at the active site.

20 glucose metabolism and deleterious effects on pancreatic βcells, widely used organophosphate pesticides might be an additional factor contributing to the development of diabetes. This proposal is to study the cytotoxicity of CPF on pancreatic βcells and possible effects on their function, insulin secretion.

The RINm5f (RIN) cell line was chosen as a model cell line to study the effects of

CPF on pancreatic βcells. RIN cell line derives from a rat tumor pancreatic islet. It secretes insulin and its insulin secretory response is similar to normal islets. Many secretagogues have been reported to stimulate insulin secretion in RIN cells, including glucose, potassium, glucagon and calcium (Bhathena et al. 1984; Chen et al. 1994;

Santangelo et al. 2007; Mao et al. 2009). For the studies presented here, we used glucose and potassium as secretagogues.

Hypothesis:

Chlorpyrifos causes cytotoxicity in insulin producing RINm5f βcells by induction of cell apoptosis and by interfering with insulin secretion and maturation through inhibition of PC enzyme activity.

Specific Aim 1: Test the hypothesis that chlorpyrifos induces RINm5f cell apoptosis.

Specific Aim 2: Test the hypothesis that chlorpyrifos interferes with insulin secretion and maturation through inhibition of PC enzyme activity.

Materials and Methods

Chlorpyrifos Preparation

CPF was prepared in acetonitrile at 200 mM and serial dilutions made from 100 mM to 5 mM, with a final concentration of 5200 µM CPF in 0.1% acetonitrile.

Cell Culture Medium

RIN cells were grown in RPMI 1640 containing 10% fetal bovine serum (FBS):

RPMI 1640 medium with 2 mM Lglutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate and 10% FBS.

On the day of the experiment(s), RIN cells were incubated with CPF in the presence of serum free media RPMI 1640 containing 0.1% bovine serum albumin

(BSA): RPMI 1640 medium with 2 mM Lglutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate and 0.1% (w/v) BSA.

Cell Culture

RINm5f (RIN) cells were obtained from ATCC. Cells were maintained at 37°C in 5%

CO 2 incubator. Medium was changed every 3 days. When cells reached 80% confluence, cells were split with 0.25% (w/v) Trypsin0.53 mM EDTA solution and plated in new T75 flasks.

MTT Assay

We utilized the Vybrant® MTT Cell Proliferation Assay Kit from Invitrogen to quantify cell viability. This assay measures the reduction of yellow

3(4,5dimethythiazol2yl)2,5diphenyl tetrazolium bromide (MTT) by mitochondrical succinate dehydrogenase to form an insoluble, dark purple formazan

23 product. The cells are then solubilized with an organic solvent i.e., 100% DMSO. The released solubilized formazan is then measured spectrophotometrically. Levels of activity reflect the viability of the cells since MTT reduction can only occur in metabolically active cells. The MTT assay is widely used for drug action, cytotoxic agents and screening other biologically active compounds.

For determination of CPF on RIN cell viability, RIN cells (5 x10 4) were seeded in

96 well plate and maintained in RPMI 1640 medium containing 10% FBS until cells reached 80% confluence. The medium then was replaced with RPMI 1640 medium containing 0.1% BSA. Cells were treated with chlorpyrifos at 0 (0.1% acetonitrile as a control), 5, 10, 25, 50, 100 and 200 µM for 2, 4 and 16 hr at 37°C in a 5% CO 2 incubator.

MTT (10 µl) was added followed by incubation for 4 hr. Then 100 µl DMSO was added and mixed thoroughly with the cells. The absorbance was measured at 570 nm.

Cell Apoptosis Assay

CPF inducedapoptosis in RIN cells was detected by a Cell Death Detection

ELISA Kit from Roche Applied Science. Apoptosis is the most common form of eukaryotic cell death. It is characterized by membrane blebbing, cytoplasm condensation and DNA fragmentation by activated endogenous endonuclease. The cleavage of double stranded DNA appears at the internucleosomal linker region and generates mono and oligonucleosomes. The capture of the mono and oligonucleosomes in the cytoplasm of the apoptotic cell is due to the fact the DNA fragmentation occurs before plasma membrane breakdown. This experiment was a quantitative determination of cytoplasmic histoneassociated DNA fragments (mono

24 and oligonucleosomes).

The detection has several stages: first, antihistone antibodies were fixed on the wall of a microplate; second, nonspecific binding sites were saturated by treatment with incubation buffer (blocking buffer); third, nucleosomes contained in the sample were bound to the immobilized antihistone antibody via their histone components; fourth, antiDNAperoxidase (POD) that reacts with the DNA of the nucleosome was added; fifth, unbound peroxidase conjugate was removed by washing; sixth, the amount of peroxidase retained in the immunocomplex was determined by reacting with ABTS (2,2azinodi[3ethylbenzthiazoline sulfonate]) as a substrate (Figure 9).

FIG. 9. Components of Cell Death Detection ELISA

Cells were seeded in 24well plates at 1X10 5 per well and maintained in RPMI

1640 medium containing 10% FBS until cells reached 80% confluence. Medium was then replaced with RPMI 1640 medium containing 0.1% BSA. Cells were treated with chlorpyrifos at 0 (0.1% acetonitrile as a control), 5, 10, 25, 50, 100 and 200 µM for 2, 4 25 and 16 hr at 37°C in a 5% CO 2 incubator. Both cells (by cell scrapper) and medium were then transferred to new tubes and centrifuged at 200 x g for 5 min. The supernatant was discarded and the cell pellet was resuspend in 1 ml culture medium followed by centrifugation at 1500 x g for 1 min. The cell pellet was resuspended with 500 µl incubation buffer and the mixture incubated for 30 min at room temperature. The lysate then was centrifuged at 20,000 x g for 10 min, after which 400 µl of the supernatant

(cytoplasmic fraction) was carefully removed and saved. The supernatant was diluted

1:10 with incubation buffer. Supernatant (100 µl) was added into MPmodules precoated with antihistone. The MPmodules were covered tightly with adhesive cover foil and incubated for 90 min at room temperature. The solution then was removed thoroughly and the wells were rinsed three times with 300 µl washing solution per well.

The washing solution was removed carefully followed by adding 100 µl of conjugate solution into each well, except the blank position. MPmodules were tightly covered with adhesive cover foil and incubated for 90 min at room temperature. The solution then was removed and the wells were rinsed 3 times with 300 µl washing solution per well. After the wells were empty, substrate solution (100 µl) was added into each well.

The MPmodules were then incubated on a shaker for about 15 min until a green color developed and measured using spectrophotometer at 405 nm against substrate solution as a blank.

This apoptosis method is very sensitive. The exact detection limit in different cells is dependent on the kinetics of cell death and the agent used to induce cell death. For example, in HL60 cells, treated with 04 µg/ml camptothecin (CAM) for 4 hr, the

26 immunoassay allows the detection of monoand oligonucleosomes in the cytoplasmic fraction of 50 cells.

Cell necrosis assay

Necrosis is characterized by increased ion permeability of the plasma membrane leading to cell swelling and rupturing the plasma membrane. RIN cell death will also be evaluated with a Cytotoxicity Detetion Kit for LDH from Roche Applied Science. It is a nonradioactive alternative to the [ 3H]thymidine release assay and the [ 51 Cr]release assay based on measurement of LDH activity released from plasma membranedamaged cells.

RIN cells were adjusted to 5 x 10 4/200 µl with RPMI medium containing 0.1%

BSA in 96 well plates. No cells were added to wells for background control and substance (CPF) control. The cells were incubated overnight in an incubator at 37°C in

5% CO 2 to allow the cells to adhere tightly. The next day, the assay medium was removed from the cells to remove LDH activity released from the cells and was replaced with 200 µl fresh medium containing 0, 5, 10, 25, 50, 100 or 200 µM CPF. For background control, 200 µl assay medium was added; for high control, 100 µl medium containing 200 µM CPF and 100 µl 2% Triton X100 were added; for substance control,

200 µl medium containing 200 µM CPF was added (Table 2). After incubation for 2, 4 and 16 hr, 100 µl/well supernatant was carefully removed and transferred into a new

96well flat bottom microplate. Reaction mixture 100 µl was added to each well and incubated for 30 min at room temperature (protecting from light). The LDH activity then was measured by a plate reader at 490 nm.

Table 2 LDH Experiment Design

Cells volume in Triton X100 the Contents of the soluction (2% in Test substance Assay medium corresponding well assay medium) (200 µM CPF) µl 0.1% acetonitrile µl medium µl Background 200 control

Low control 200

High control 100 100

Substance 200 control 5 µM CPF 200 treatment 10 µM CPF 200 treatment 25 µM CPF 200 treatment 50 µM CPF 200 treatment 100 µM CPF 200 treatment 200 µM CPF 200 treatment

Assay medium: 0.1% acetonitrile RPMI medium containing 0.1% BSA

Background control: 200 µl assay medium to triplicate wells

Low control: spontaneous LDH release

High control: maximum LDH release

Cell toxicity by necrosis was calculated as follows:

Cytotoxicity (%)=(exp. value low control)/ (high control low control)*100

Insulin Secretion

RIN cells (1X10 5) were seeded in 24well plates and maintained in RPMI 1640 medium containing 10% FBS until cells reach 80% confluence. The medium then was replaced with RPMI 1640 medium containing 0.1% BSA. Cells were treated with chlorpyrifos for 2, 4 and 24 hr. Cells then were washed with KRBH buffer ( 0.1% BSA,

125 mM NaCl, 4.74 mM KCl, 1 mMCaCl 2, 1.2 mM KH2PO 4, 1.2 mM MgSO 4, 5 mM

NaHCO 3 and 25 mM HEPES, pH 7.4) three times. Then basal insulin secretion was analyzed by incubation with KRBH buffer containing 2.8 mM glucose for 0.5 hr. After collecting the medium, KRBH buffer containing either 25 mM glucose or 50 mM potassium chloride was incubated for 1 hr. The stimulated insulin medium was then collected for detection. The cells then were lysed with 300 µl 0.1 N HCl and were transferred to a new tube followed by centrifugation at 13,000 rpm for 20 min at 4°C and supernatant collection. Basal, stimulated and extracted cell protein were stored at

80°C until use.

Insulin Detection by ELISA assay

The basal, stimulated and extracted RIN cell proteins were all analyzed for insulin using an ALPCO rat insulin ELISA kit. The assay is a sandwich type immunoassay.

Monoclonal antibodies specific for insulin are immunoblized to the 96well microplate.

After standards, controls and samples containing insulin are added to the wells, another insulin antibody which is labeled with a horseradish peroxidase enzyme (Conjugate) is added. As a result, insulin molecules are sandwiched between the solid phase of the plate and the Conjugate. After incubation and washing, 3,3´,5,5´tetramethylbenzidine

(TMB) which is the substrate of the horseradish peroxidase, is added to show the blue color. The intensity of the color generated is directly proportional to the amount of insulin in the samples. Stop solution then is added to stop the reaction and change the color from blue to yellow.

Insulin standard (10 µl), reconstituted control (10 µl) and the samples (10 µl) were added into their respective wells followed by adding 75 µl of working strength conjugate into each well. The plate was then incubated for 2 hr at room temperature on a Belly Dancer (Stovall, Inc. Greensboro, N.C.). The microplate was then washed with wash buffer for 6 times. TMB substrate 100 µl was added into each well and incubated for 15 min on the Belly Dancer. Stop solution 100 µl then was added to stop the reaction followed by reading the absorbance at 450 nm. The amount of insulin in the samples was calculated based on the plotted standard curve. The unit of detected insulin was ng/ml.

Proinsulin Detection by ELISA assay

Proinsulin detection was the same as insulin detection by the ELISA assay, except that proinsulin antibodies were coated to the 96well microplate. The assay is very sensitive with the detection limit as low as several pmol/L (pM).

Western Blotting Analysis for Prohormone Convertase 1 and 2

Western Blotting was used to detect prohormone enzyme PC1 and PC2 expression affected by CPF. RIN cells treated with various CPF concentrations for 4 hr were lysed with lysis buffer containing 20 mM Tris HCl pH 8, 137 mM NaCl, 10% glycerol, 1% glycerol, 1% NP40 and 2 mM EDTA. The lysates were collected and homogenized

30 followed by centrifugation at 4°C and 13,000 RPM for 15 min. Supernatants were collected for protein determination by Bradford method using Biorad protein assay. As the protein concentrations were very low, Ultacel YM membrane filter, YM30 from

Millipore was used to concentrate the proteins. The microcon sample reservoir was inserted into their corresponding vial and the sample proteins were added into the sample reservoir. The attached cap was sealed and centrifuged 14,000 g for 24 min at

4°C. The vial and sample reservoir was then separated. The sample reservoir was placed upside down in a new vial and centrifuged 3 min at 1,000 g to transfer concentrate to the vial. The concentrated proteins were then analyzed by the Biorad protein assay. One hundred µg proteins of each sample were loaded onto a 1020%

TrisHCl Biorad Criterion SDS gel. The separation conditions were 150 V for 90 min.

After separation, the proteins were transferred to a Citerion Gel PVDF blotting membranes overnight at 18 V and 100 mA. Upon completion, the PVDF membranes were blocked in Amersham membrane blocking agent (1.5 g blocking agent in 50 ml

1X PBS and 0.1% Tween20 (PBST)) for 1 hr with gentle shaking. Membranes were then incubated with either rabbit antiprohormone convertase 1 (1:1000) (Millipore

Corporation) or rabbit antiprohormone convertase 2 (1:50) (Millipore Coorporation) primary antibodies in blocking agent overnight at 4°C. Upon completion of primary antibody incubation, the PDVF membranes were washed in wash buffer 3 times followed by addition of the secondary antibodies conjugated to horseradish peroxidase in wash buffer (1: 20,000) for 1 hr. Following the wash, membranes were incubated with the enhanced chemiluminescence (ECL) substrate (Pierce) and imaged using a

Fuji FLA5100 PhosphorImager and Image Guage software.

Detection of Prohormone Convertase 2 Activity

PC2 enzyme activity was determined using a fluorescent substrate. PC enzymes are able to cleave at paired basic amino acids site at the appropriate pH and ion concentration. Upon cleavage, a fluorescent compound is released and detected by fluorescence spectrophotometer at 360 nm excitation and 480 nm emission wavelengths. The intensity of the released fluorescence is in proportion to PC enzyme activity. In order to analyze a specific PC enzyme activity, an inhibitor of this enzyme is added in parallel incubations. The PC enzyme activity then is calculated by subtracting the activity with the inhibitor from the total PC enzyme activity. Currently, the only available inhibitor is the PC2 enzyme inhibitor. The inhibitor is a peptide having the same amino acid sequence of 7B2 Cterminal peptide (CT peptide),

SVNPYLQGKRLDNVVAKK.

RIN cells 1x10 6 were treated with CPF for 4 hr followed by protein extraction in

200 µl lysis buffer (50 mM TrisHCl pH 7.5 containing 1% Triton X100, 10% glycerol and protease inhibitor cocktail (SigmaAldrich). After homogenization, the lysate was centrifuged at 13,000 RPM for 15 min at 4°C. Supernatants were collected and protein concentrations were determined. Sample aliquots were stored at 80°C until further use.

Total proteins (30 µg) from cells were incubated with 100 µM

LpGluArgThrLysArg7amino4methylcoumarin (Peptide International) in reaction buffer (100 mM sodium acetate, pH 5.0 and 2 mmol/L CaCl2 ) at 37°C for 100 min. In parallel, 1 µM CT peptide SVNPYLQGKRLDNVVAKK (dissolved in water,

Synthesized by Biomatik), a PC2specific inhibitor (Zhu et al, 1996), was added to the reactions. A fluorescence spectrophotometer was used to monitor the release of

7amino4methylcoumarin (AMC) every 10 min up to 50 min. The activity inhibited by the presence of CT peptide was considered as PC2specific activity.

The PC2 activity was calculated as follow,

PC2 activity=Fluorescence released from the substrate incubation without CT peptidefluorescence release from the substrate incubation with CT peptide.

PC2 Activity Detection by MALDITOF MS Analysis

Matrixassisted laser desorption/ionisationtime of flight mass spectrometry

(MALDITOF MS) is a novel technique in which a matrix and biomolecules such as proteins, peptide are irradiated by laser pulses. The matrix absorbs most of the laser energy to prevent unwanted fragmentation of the biomolecules. The ionized biomolecules are accelerated in an electric field and enter the flight tube. During the flight in this tube, different molecules are separated according to their mass to charge ratio and reach the detector at different times. Therefore, each molecule yields a distinct signal based primarily on the mass. The method is used for detection and characterization of biomolecules with molecular masses between 400 and 350,000

Dalton. It is a very sensitive method, which allows the detection of low (10 15 to 10 18 mole) quantities of sample with an accuracy of 0.1 0.01 %.

The reactions used for detecting fluorescent substrate cleavage by PC2 were used.

Matrix of 1 µl, 10 mg/ml 2, 5dihydroxy benzoic acid (DHB) (60% acetonitrile, 10% acetone and 0.3% Trifluoroacetic acid TFA), was mixed with 1 µl reactions and loaded on the MALDITOF plate. After the samples dried, the samples were analyzed using a

Bruker MALDITOF MS with reflector mode. For each sample, 2000 accumulated shots were collected.

Inhibitor Study for Apoptosis and Necrosis

As 50 µM of CPF at 4 hr showed more apoptosis and less necrosis on RIN cells, 50

µM and 4 hr exposure was chosen to study the possible roles of oxidative stress, p38,

JNK on RIN cell death. Different concentrations of antioxidant NAcetylLcysteine

(NAC), p38 inhibitor SB202190 and JNK inhibitor SP600125 were preincubated with

RIN cells for 30 min.

For the NAC studies, 200 mM, 100 mM and 50 mM stock NAC (1000 times as final concentrations) were prepared in PBS. Experiment groups were set as below

(Table 3).

Table 3 Antioxidant NAC Experimental Design

NAC __ + __ + + + (concentrations) (0.1%PBS) (200 µM) (0.1%PBS) (50 µM) (100 µM) (200 µM)

CPF __ __ + + + + (concentrations) (0.1%acetonitirle) (0.1%acetonitrile) (50 µM) (50 µM) ( 50 µM) (50 µM)

For p38 inhibitor studies, 5 mM, 10 mM and 20 mM SB202190 (1000 times as

34 final concentrations) were prepared in ethanol. Experiment groups were set as below

(Table 4).

Table 4 P38 Inhibitor Experimental Design

SB202190 __ + __ + + + (concentrations) (0.1%ethanol) (20 µM) (0.1%ethanol) (5 µM) (10 µM) (20 µM) CPF __ __ + + + + (concentrations) (0.1%acetonitirle) (0.1%acetonitrile) (50 µM) (50 µM) ( 50 µM) (50 µM)

For JNK inhibitor studies, 5 µM, 10 µM and 20 µM SP600125 were prepared as final concentrations in DMSO. Experiment groups were set as below (Table 5).

Table 5 JNK Inhibitor Experimental Design

SP600125 __ + __ + + + (concentrations) (0.1% DMSO) (20 µM) (0.1%DMSO) (5 µM) (10 µM) (20 µM)

CPF __ __ + + + + (concentrations) (0.1%acetonitirle) (0.1%acetonitrile) (50 µM) (50 µM) ( 50 µM) (50 µM)

Note: the concentrations in parentheses were final concentrations.

Following the treatment, apoptosis and necrosis experiments were performed.

Results

Specific Aim 1

Cytotoxicity detection by MTT

To determine CPF cytotoxicity, RIN cells were treated for 2, 4 and 16 hr with various concentrations of CPF or with the acetonitrile vehicle (0.1% final concentration). Cell viability was analyzed by using the MTT assay. CPF caused dose and timedependent reduction RIN viability at concentrations greater than 50 µM at 2 hr, 4 hr and 16 hr (Figure 10). The results suggest that CPF induces RIN cell cytotoxicity.

120

100

** 80

** 2 hours 60 4 hours ** 16 hours 40 ** **

Percent cell Percent viability ** 20 ** ** **

0 0 5 10 25 50 100 200 CPF (µM)

FIG. 10. CPF decreases MTT metabolism in RIN cells. RIN cells were treated with

CPF at 0 (0.1% acetonitrile as a control), 5, 10, 25, 50, 100 and 200 µM for 2, 4 and 16 hr. The cells were incubated with MTT for 4 hr followed by incubation with DMSO.

The absorbance was measured at 570 nm. Results were expressed as percent cell viability. Data is representative of three independent experiments. Statistical analysis was performed using TwoWay ANOVA. Bonferroni test was used for Posthoc comparison. N=3 ** denotes p< 0.01 vs. control.

Cell apoptosis Assay

To determine whether apoptosis was responsible for the decrease in RIN cell viability, we measured markers of apoptotic cell death. The results indicated that CPF, caused a progressive, doserelated increase in apoptosis with respect to the untreated controls as measured at 4 and 16 hr postexposure (Figure 11). CPF at ≥ 50 µM increased apoptosis ≈4 fold after 4 and 16 hr treatment. In contrast, 2 hr postexposure to 50 µM CPF did not induce a significant increase in apoptosis. Two hr exposure to

100 and 200 µM did produce a significant effect.

2 hr

10 8 ** ** 6

4 2 0 Apoptosis (FoldChange)Apoptosis 0 5 10 25 50 100 200 CPF (µM) 4 hr

10 8 ** 6 ** ** 4 2 0 Apoptosis (FoldApoptosis Change) 0 5 10 25 50 100 200 CPF (µM) 16 hr

10 ** 8 6 ** ** 4 2 Apoptosis (FoldApoptosis Change) 0 0 5 10 25 50 100 200 CPF (µM) FIG. 11. CPF induces RIN cell apoptosis. RIN cells were treated with CPF at 0 (0.1% acetonitrile as a control), 5, 10, 25, 50, 100 and 200 µM for 2, 4 and 16 hr. The Cell

Death ELISA Kit was used for detecting apoptosis. Results were expressed as fold of control. The values are expressed as the mean ±SEM. Data is representative of three independent experiments. Statistical analysis was performed using TwoWay ANOVA.

Bonferroni test was used for Posthoc comparison.

** denotes p< 0.01 vs. control. 39

Cell necrosis assay

In addition to apoptosis, necrosis is another type of cell death. The apoptosis experiment showed that CPF could induce apoptotic cell death. However, there is no reason to exclude that RIN cells could die through necrosis. LDH released from the plasma membrane was used as a test for necrosis. The occurrence of necrotic cell death appeared to be at 50 µM CPF and was time and dosedependent (Figure 12). At 50 µM, necrosis increased in RIN cells from 8.9 ±1.3% at 2 hr to 17.14 ±6.9% at 4 hr and

20.81 ±1.6% at 16 hr in comparison to control (Figure 12).

(A)

2 hr

30 25

20 ** 15 ** 10 ** 5 0 0 5 10 25 50 100 200 CPF (µM)

(B) 4 hr

30 25 ** ** 20 ** 15 10 5 0 0 5 10 25 50 100 200 CPF (µM) (C)

16 hr

30 ** 25 ** 20 ** 15 10

Cell Death (%) 5 Cell Death (%) Cell Death (%) 0 0 5 10 25 50 100 200 CPF (µM)

FIG. 12. CPF induces necrotic cell death in RIN cells. RIN cells were treated with CPF at 0 (0.1% acetonitrile as a control), 5, 10, 25, 50, 100 and 200 µM for 2 (A), 4 (B) and

16 hr (C). Cellular necrosis was determined using the Roche LDH kit. Results were expressed as percent cell death. The values are expressed as the mean ±SEM. Data represents of three independent experiments. Statistical analysis was performed using

TwoWay ANOVA. Bonferroni test was used for Posthoc comparison.

** Denotes p< 0.01 vs. control.

The percentage of apoptosis and necrosis of RIN cells treated with CPF

Based on the results, CPF induces two types of cell death, apoptosis and necrosis.

In order to determine the proportion of each type of cell death, we made the assumption that cell viability assay (MTT experiment) accurately reflects cell death. Table 6 summarizes the types of cell death and their corresponding percentage in the whole population. The table indicates that the majority of cell death was due to apoptosis but necrosis also contributed to a significant amount of cell death.

Table 6 Comparison of cell death affected by CPF

CPF concentration 50 100 200

Cell death type A N A N A N

2 hr 7.89 8.90 47.33 14.50 44.31 18.70

4 hr 24.65 17.14 43.98 20.39 45.22 20.09

16 hr 54.04 20.81 56.07 22.77 56.38 27.43

“A” represents apoptosis whereas “N” represents necrosis.

The numbers represent a percentage of the total number of cells counted, both alive and dead. Thus the percent live cells would be the difference between total cell counts minus apoptotic + necrotic cell counts.

Antioxidant NAcetylLcysteine (NAC) on Cell Viability

To explore the potential mechanisms of CPF induced cell death, inhibitors were used to investigate their effects on RIN cell death. The mechanism of apoptosis has been well studied for a long time, whereas very limited work has been done on the mechanism of necrotic cell death. We chose a CPF concentration and exposure duration that would cause more RIN cell death by apoptosis and less by necrosis. As such, further characterization of CPF toxicity was performed using 50 µM CPF and 4 hr exposure. It is well demonstrated that pesticides could cause the production of oxidative stress. We then tested whether an oxidative stress inhibitor, i.e. NAC, could suppress cell death, either apoptosis or necrosis. In order to determine the specificity of

NAC on the alteration of either type of cell death, we used 3 different concentrations of

NAC (50, 100 and 200 µM). Our results suggest that NAC had dosedependent protection of CPFinduced RIN apoptosis. At 200 µM, NAC significantly inhibited

CPFcaused cell apoptosis by 22.5% (Figure 13). When the effect of NAC on

CPFinduced necrosis was tested, there was no significant influence at any concentration. NAC itself induced increased RIN cell necrotic cell death and did not exhibit a protective effect on CPFinduced necrosis (Figure 14). Our results indicate that antioxidant NAC may specifically protect RIN cells from injury associated via apoptosis, but not necrosis as induced by CPF.

* 3

Apoptosis (fold change) (fold Apoptosis 0 NAC 200 M 50 M 100 M 200 M CPF + + + +

FIG. 13. The effects of antioxidant NAcetylLcysteine (NAC) on CPFinduced RIN cell apoptosis. The RIN cells were pretreated with PBS (vehicle for NAC, column 1 and column 3), 200 µM NAC (column 2), and 3 different concentrations of antioxidant

NAcetylLcysteine (NAC) (50, 100 and 200 µM; column 4, 5 and 6 respectively) for

30 min followed by acetonitrile (vehicle for CPF, column 1 and column 2) or 50 µm

CPF treatment (column 3, 4, 5, and 6) for 4 hr. The Cell Death ELISA kit was used for detecting apoptosis. Results were expressed as fold of control. The values are expressed as the mean ±SEM. Data is representative of three independent experiments. Statistical analysis was performed using OneWay ANOVA. Tukey test was used for Posthoc comparison. * Denotes p<0.05 compared to CPF only group (column 3).

Cell (%) death Cell 5

0 NAC 200 µM 50 µM 100 µM 200 µM CPF + + + +

FIG. 14. The effects of antioxidant NAcetylLcysteine (NAC) on CPFinduced RIN cell necrosis. The cells were pretreated with PBS (vehicle for NAC, column 1 and column 3), 200 µM NAC (column 2), and 3 different concentrations of antioxidant

NAcetylLcysteine (NAC) (50, 100 and 200 µM; column 4, 5 and 6 respectively) for

30 min followed by acetonitrile (vehicle for CPF, column 1 and column 2) or 50 µm

CPF treatment (column 3, 4, 5, and 6) for 4 hr. Cellular necrosis was determined using the Roche LDH Kit. Results were expressed as percent cell death. The values are expressed as the mean ±SEM. Data is representative of three independent experiments.

Statistical analysis was performed using OneWay ANOVA.

Inhibition of p38 and JNK MAPK Kinases on Cell Viability

Activation of cJun Nterminal kinases/stressactivated protein kinase (JNK/SAPK,

JNK) and p38 MAPK is associated with apoptosis, and inhibition of their activation rescues cells from apoptotic death (Chang et al. 2001; Cai et al. 2006; Zhou et al. 2006;

Tanel et al. 2007). To ascertain whether JNK and p38 pathway mediated CPFinduced apoptosis and necrosis, RIN cells were exposed to CPF in the presence and absence of 2 inhibitors, the p38 inhibitor SB202190, or the JNK inhibitor SP600125, for 4 hr. The mechanistic pathways were explored briefly using two inhibitors. For the p38 inhibitor,

SB202190, the concentrations used were 5, 10 and 20 µM. For the JNK inhibitor,

SP600125, 5, 10 and 20 µM were used. The p38 inhibitor SB202190 at 5, 10 and 20 µM significantly reduced CPFinduced apoptosis 1.28, 1.68 and 2.46 fold respectively

(Figure 15). However, SB202190 failed to inhibit the necrosis effects of CPF (Figure

16). JNK inhibitor SP600125 also significantly reversed CPFinduced apoptosis 2.12 and 2.27 fold at 10 and 20 µM respectively (Figure 17). In contrast to SB202190,

SP600125 also showed a protective effect on CPFinduced cell necrosis. Cell necrosis reduced from 15.04% to 9.6% and 10.28% respectively at 10 and 20 µM concentrations

(Figure 18). These results indicated that p38 mediates pathways which may induce RIN cell apoptosis, but not necrosis. JNK mediates pathways which induce not only RIN cell apoptosis, but also necrosis.

* 3 **

2 **

1 Apoptosis (fold change) (fold Apoptosis

0 SB202190 20 M 5 M 10 M 20 M CPF + + + + FIG. 15. The p38 inhibitor SB202190 inhibits CPFinduced RIN cell apoptosis. The cells were pretreated with ethanol (vehicle for SB202190, column 1 and 3), 20 µM

SB202190 (column 2), and 3 different concentrations of SB202190 (5, 10 and 20 µM; column 4, 5 and 6 respectively) for 30 min followed by acetonitrile (vehicle for CPF, column 1 and column 2) or 50 µM CPF treatment (column 3, 4, 5, and 6) for 4 hr. The

Cell death ELISA Kit was used for detecting apoptosis. Results were expressed as fold of control. The values are expressed as the mean ±SEM. Data is representative of three independent experiments. Statistical analysis was performed using OneWay ANOVA.

Tukey test was used for Posthoc comparison. * Denotes p<0.05 and ** denotes p< 0.01 vs. CPF only group.

Cell death (%) death Cell 5

0 SB202190 20 M 5 M 10 M 20 M CPF + + + +

FIG. 16. The effects of p38 inhibitor SB202190 on CPFinduced RIN cell necrosis. The cells were pretreated with ethanol (vehicle for SB202190, column 1 and column 3), 20

µM SB202190 (column 2), and 3 different concentrations of SB202190 (5, 10 and 20

µM; column 4, 5 and 6 respectively) for 30 min followed by acetonitrile (vehicle for

CPF, column 1 and column 2) or 50 µM CPF treatment (column 3, 4, 5, and 6) for 4 hr.

Cellular necrosis was determined using the Roche LDH Kit. Results were expressed as percent cell death. The values are expressed as the mean ±SEM. Data is representative of three independent experiments. Statistical analysis was performed using OneWay

ANOVA.

2 ** **

Apoptosis (fold change) (fold Apoptosis 0 SP600125 20 M 5 M 10 M 20 M CPF + + + +

FIG. 17. The JNK inhibitor SP600125 inhibits CPFinduced RIN cell apoptosis. The cells were pretreated with DMSO (vehicle for SP600125, column 1 and column 3), 20

µM SB202190 (column 2), and 3 different concentrations of SP600125 (5, 10 and 20

µM; column 4, 5 and 6 respectively) for 30 min followed by acetonitrile (vehicle for

CPF, column 1 and column 2) or 50 µM CPF treatment (column 3, 4, 5, and 6) for 4 hr.

The Cell Death ELISA Kit was used for detecting apoptosis. Results were expressed as fold of control. The values are expressed as the mean ±SEM. Data is representative of three independent experiments. Statistical analysis was performed using OneWay

ANOVA. Tukey test was used for Posthoc comparison. ** Denotes p< 0.01 vs. CPF only group (column 3).

15 * * 10

Cell death (%) Cell 5

0 SP600125 20 M 5 M 10 M 20 M CPF + + + +

FIG. 18. The JNK inhibitor SP600125 inhibits CPFinduced RIN cell necrosis. The cells were pretreated with DMSO (vehicle for SP600125, column 1 and column 3), 20

µM SB202190 (column 2), and 3 different concentrations of SP600125 (5, 10 and 20

µM; column 4, 5 and 6 respectively) for 30 min followed by acetonitrile (vehicle for

CPF, column 1 and column 2) or 50 µM CPF treatment (column 3, 4, 5, and 6) for 4 hr.

ANOVA. Tukey test was used for Posthoc comparison. * Denotes p< 0.05 vs. CPF only group (column 3).

Specific Aim 2

Basal insulin and proinsulin

Insulin secretion reflects βcell function. Proinsulin is the prohormone precursor of insulin. This specific aim focused on βcell secretion affected by CPF at lower concentrations than lethal doses. Three different time points (2, 4, 24 hr) and various

CPF concentrations (0, 5, 10, 25 µM) were chosen to evaluate basal secretion of insulin

(in the presence of 2.8 mM glucose). After 2, 4 and 24 hr incubation with different concentrations of CPF, basal insulin secretion was unchanged compared to control cells

(Figure 19). Basal proinsulin secretion was detected at 4 hr postexposure to CPF.

There was no significant change at any doses (Figure 20).

0.06

0.05

0.04 2 hr 4 hr 0.03 24 hr Insulin (ng/ml/min) 0.02

0.01 0 5 10 25 CPF (µM)

FIG. 19. Effect of CPF on basal insulin secretion. Cells were treated with 0 (0.1%

acetonitrile as control), 5, 10 and 25 µM CPF for 2, 4 and 24 hr. Cells were then washed

with KRBH buffer three times. Basal insulin secretion was detected by incubating

KRBH buffer containing 2.8 mM glucose for 0.5 hr. The level of insulin in the culture

medium was analyzed by ELISA and normalized to ng/ml/min. Data represents three

independent experiments. Statistical analysis was performed using TwoWay ANOVA.

1 Proinsulin (pmol/L/min) Proinsulin 0 0 5 10 25

CPF (µM) FIG. 20. Effect of CPF on basal proinsulin secretion. RIN cells were treated with 0 (0.1% acetonitrile as control), 5, 10 and 25 µM CPF for 4 hr. Cells were then washed with

KRBH buffer three times. Basal proinsulin secretion was detected by incubating KRBH buffer containing 2.8mM glucose for 0.5 hr. The level of proinsulin in the culture medium was analyzed by the ELISA normalized to pmol/L/min. Data represents three independent experiments. Statistical analysis was performed using OneWay ANOVA.

Stimulated Insulin and Proinsulin Secretion by Potassium

To verify whether CPF could cause a functional impairment of insulin secretion in

RIN cells, CPFtreated RIN cells were stimulated with a depolarizing concentration of

KCl (50 mM) for 1 hr following basal secretion. Insulin and proinsulin were both analyzed. Our data showed that noncytotoxic concentrations of CPF (10 and 25 µM) significantly inhibited RIN cells insulin secretion in a dose and timedependent manner at 4 hrand 24 hr (Figure 21). At concentrations as low as 5 µM, CPF inhibited insulin secretion at 4 hr as well. Potassiumstimulated insulin secretion was 4 to 11fold more than the basal insulin secretion (Figure 21 and Table 7). If the ratio of stimulated secretion to basal secretion is greater than one, then the secretory pathway is called a regulated secretory pathway; if the ratio is equal or less than one, the secretory pathway is called a constitutive secretory pathway. Our results verify that potassiumstimulated insulin secretion is through the regulated secretory pathway (Table 7). CPF has an adverse effect on regulated secretion of insulin in RIN cells at concentrations of 10 and

25 µM and exposure times of 4 and 24 hr (Table 7). In contrast, RIN cells showed a significant reduction in potassiumstimulated proinsulin secretion to that of basal proinsulin at all concentrations (Figure 22). However after 4 hr treatment with CPF, there was significant increase in proinsulin secretion compared to control cells

(stimulated proinsulin). Potassiumstimulated proinsulin was 0.655, 0.809 and 0.814 fold of basal proinsulin (Table 8) which qualifies a constitutive secretory pathway.

0.5

0.4

2 hr 0.3 4 hr

* 24 hr 2 hr basal 0.2 ** ** 4 hr basal ** ** 24 hr basal Insulin(ng/ml/min) 0.1

0 0 5 10 25 CPF (µM) FIG. 21. Effect of CPF on potassiumstimulated insulin secretion. RIN cells were

treated with various CPF concentrations for 2, 4 and 24 hr. After collecting basal insulin,

cells were then stimulated with KRBH buffer containing 50 mM potassium for 1 hr,

followed by analyzing secreted medium. The level of stimulatedinsulin in the culture

medium was analyzed by ELISA and normalized to ng/ml/min. Basal insulin secretion

from Figure 19 was drawn for comparison. Data represents three independent

experiments. Statistical analysis was performed using TwoWay ANOVA. Bonferroni

test was used for Posthoc comparison. * p< 0.05 and ** p< 0.01 vs. control.

Table 7 Insulin stimulation by potassium

CPF concentration 0 5 10 25

2 hr 11.135±0.929 8.782±1.366 7.805±0.975 8.681±2.446

CPF exposure time 4 hr 11.017±1.239 8.077±1.343 6.484±0.309* 6.534±0.374**

24 hr 10.431±1.171 6.903±1.100 4.619±0.484* 4.197±0.731**

Insulin Stimulation by Potassium = (stimulated/unstimulated)

Notes: Insulin secretion unit is ng/ml/ min. Statistical analysis was performed using

TwoWay ANOVA. Bonferroni test was used for Posthoc comparison. * Represents p<0.05 and ** represents p<0.01 compared to control group (0).

* * * stimulated proinsulin basal proinsulin 1 Proinsulin Proinsulin (pmol/L/min)

0 0 5 10 25 CPF (µM)

FIG. 22. Effect of potassiumstimulated proinsulin secretion. RIN cells were treated

with 0 (0.1% acetonitrile as control), 5, 10 and 25 µM CPF for 4 hr. After collecting

basal proinsulin, cells were then stimulated with KRBH buffer containing 50 mM

potassium for 1 hr, followed by analyzing secreted medium. The level of

stimulatedproinsulin in the culture medium was analyzed by ELISA and normalized to

pmol/L/min. Basal proinsulin (4 hr) from Figure 20 was drawn for comparison. Data

represents three independent experiments. Statistical analysis was performed using

OneWay ANOVA. Tukey test was used for Posthoc comparison. * Represents p< 0.05

vs. control.

Table 8 Proinsulin stimulation by potassium

CPF concentration 0 5 10 25

CPF exposure time 4 hr 0.655±0.065 0.761±0.012 0.809±0.028 0.814±0.008

Proinsulin Stimulation by potassium= (stimulated insulin/basal insulin)

Statistical analysis was performed using OneWay ANOVA.

Stimulated Insulin and Proinsulin Secretion by Glucose

Glucose is a physiological secretagogue for insulin secretion. We evaluated glucosestimulated (25 mM) insulin and proinsulin secretion after 4 hr incubation with

0, 5, 10 and 25 µM CPF following the basal insulin secretion. Our data showed that 25 mM glucose caused less insulin secretion than that of 50 mM potassium (Figure 23A and Figure 21). Glucosestimulated insulin secretion was significantly decreased in

RIN cells incubated with CPF for 4 hr, at all concentrations, compared to control

(Figure 23A) and the ratio of stimulated to basal insulin showed significant changes

(Table 9). Proinsulin secretion showed a similar pattern as 50 mM potassium stimulation, i.e. reduced proinsulin secretion to that of basal (Figure 23B). The ratio of stimulated proinsulin to basal proinsulin had an increased trend and showed significance at 25 µM (Table 10).

(A)

0.10

0.08

0.06 ** ** 4 hr ** 0.04 4 hr basal

Insulin (ng/ml/min) 0.02

0.00 0 5 10 25 CPF (µM) (B)

** 4 hr * 4 hr basal 1 Proinsulin Proinsulin (pmol/L/min)

0 0 5 10 25 CPF (µM)

FIG. 23. Effect of glucosestimulated insulin and proinsulin secretion. RIN cells were

treated with various CPF concentrations for 4 hr. After collecting basal insulin and

proinsulin, cells were then stimulated 25 mM glucose for 1 hr, followed by analyzing

secreted medium for insulin (A) and proinsulin (B). Basal insulin and proinsulin

secretion (4 hr) was drawn for comparison. The values are expressed as the mean ±

SEM from three independent experiments. Statistical analysis was performed using one

way ANOVA. * p< 0.05 and ** p< 0.01 vs. control. 61

Table 9 Insulin stimulation by glucose

CPF concentration 0 5 10 25

CPF exposure time 4 hr 2.057±0.095 1.656±0.103 1.516±0.094* 1.598±0.104*

Insulin Stimulation by Glucose = (stimulated insulin/basal insulin)

Table 10 Proinsulin stimulation by glucose

CPF concentration 0 5 10 25

CPF exposure time 4 hr 0.515±0.024 0.896±0.052 0.828±0.153 0.977±0.071*

Proinsulin Stimulation by Glucose = (stimulated proinsulin/basal proinsulin)

Notes: Insulin secretion unit is ng/ml/ min. Statistical analysis was performed using

OneWay ANOVA. Tukey test was used for Posthoc comparison. * Represents p<0.05 compared to control group (0).

Intracellular Insulin and Proinsulin After Potassium Stimulation

In order to have a complete picture of total insulin and proinsulin in the cells, intracellular insulin and proinsulin were measured. Cells were treated with various concentrations of CPF for different times followed by basal and 50 mM potassium stimulation and then cellular total protein collection.

CPFtreated cells showed no significant difference in intracellular insulin except at

25 µM and 4 hr postexposure (Figure 24). Further, intracellular proinsulin after 4 hr treatment had no significant change (Figure 25).

25 2 hr 20

5 Insulin (ng/ml) 0 0 5 10 25 CPF (µM)

20 4 hr

15 ** 10

5 Insulin (ng/ml) 0 0 5 10 25 CPF (µM)

25 20 24 hr 15 10 5 0 0 5 10 25 Insulin(ng/ml)

CPF (µM) FIG. 24. Changes of intracellular insulin after 50 mM potassium stimulation. RIN cells

were treated with various CPF concentrations for 2, 4, and 24 hr. After collecting basal

and stimulated (50 mM potassium) insulin, cells were then lysed with 0.1 N HCl

followed by protein extraction. The level of insulin was analyzed by ELISA. The values

are expressed as the mean ±SEM from three independent experiments. Statistical

analysis was performed using unpaired TwoWay ANOVA. Bonferroni test was used

for Posthoc comparison. ** Represents p<0.01 vs. control.

2500

2000

1500

1000

Proinsulin Proinsulin (pmol/L) 500

0 0 5 10 25 CPF (µM) FIG. 25. Intracellular proinsulin after 50 mM potassium stimulation. RIN cells were treated with various CPF concentrations for 4 hr. After collecting basal and stimulated

(50 mM potassium) proinsulin, cells were then lysed with 0.1 N HCl followed by protein extraction. The level of proinsulin was analyzed by ELISA. The values are expressed as the mean ±SEM from three independent experiments. Statistical analysis was performed using OneWay ANOVA.

Intracellular Insulin and Proinsulin After glucose Stimulation

To verify whether after glucose stimulation the amount of intracellular insulin and proinsulin had a similar pattern as that after potassium stimulation, RIN cells were treated with various CPF concentrations for 4 hr followed by 2.8 mM glucose (basal) and 25 mM glucose stimulation and then cellular proteins were extracted. CPFtreated

RIN cells showed decreases in intracellular insulin (Figure 26A). However, cellular proinsulin did not show any significant changes (Figure 26B).

(A)

15 * ** ** 10

0 0 5 10 25 CPF (µM)

(B)

2500

2000

1500

1000

Proinsulin (pmol/L)Proinsulin 500 Insulin (ng/ml)

0 0 5 10 25 CPF (µM)

FIG. 26. Intracellular insulin and proinsulin after glucose stimulation. RIN cells were treated with various CPF concentrations for 4 hr. After collecting basal (2.8 mM glucose) and stimulated (25 mM glucose) insulin and proinsulin, cells were then lysed with 0.1 N HCl followed by protein extraction. The level of insulin (A) and proinsulin

(B) were analyzed by ELISA. The values are expressed as the mean ±SEM from three independent experiments. Statistical analysis was performed using OneWay ANOVA.

Tukey test was used for Posthoc comparison. * Represents p< 0.05 and ** p<0.01 vs. control. 67

PC Enzyme Expression in RIN cells

Since our hypothesis links decreased insulin secretion and cellular content with inhibited PC enzymes, we next determined PC enzyme expression in RIN cells in the presence and absence of various CPF concentrations (5, 10, 25, 50, 100 and 200 µM) at

4 hr using western blotting. PC1 enzyme exhibited two bands in RIN cells (Figure

27A upper panel). The band at 164 kD represents proPC1 and the band at 115 kD represents active PC1. RIN cells showed elevated expression of PC1 at concentrations higher than 25 µM (Figure 27A 115 kDa). RIN cells treated with CPF had no change on

PC2 expression (Figure 27B). Our results indicated that PC 1 and PC 2 are expressed in

RIN cells and PC1 is upregulated by CPF.

(A)

PC 1 ProPC1 164 kDa (high molecular weight) PCActive 1 PC1 115 kDa (low molecular weight) βActinβActin 42 kDa

0 5 10 25 50 100 200 (µM) PCPC1 1 (high(164 higher kDa) molecular m olecular weight) 3

1 Fold Fold change Relative Intensity (arbitrary unit)

0 0 5 10 25 50 100 200 CPF (µM)

PC1 (low(115 molecularkDa) weight) 3 ** * **** * 2 * *

Relative Intensity (arbitrary unit) Relative Relative Intensity unit) (arbitrary Fold Fold change 0 0 5 10 25 50 100 200 CPFCPF (µM) (µM)

(B)

PC 2 101 kDa

ßActin 42 kDa 0 5 10 25 50 100 200 (µM) P C 2 wPC2 estern 1 . 5

y it ) s it 1 . 0 n n te u In y e r v ra ti it a b el r 0 . 5 R (a

Fold change 0 . 0 0 5 10 25 50 100 200 CPF (µM)

FIG. 27. PC1 and PC2 expression in RIN cells. RIN cells were treated with various CPF concentrations (0, 5, 10, 25, 50, 100, 200 µM) for 4 hr. Cells were then lysed and proteins were extracted and quantified. Aliquots of the total lysate containing an equivalent amount of protein (100 µg/lane) were used. Quantification data was expressed as arbitrary units of intensity relative to βactin. The values are expressed as the mean ± SEM from three independent experiments. Statistical analysis was performed using OneWay ANOVA. Tukey test was used for Posthoc comparison.

* Represents p< 0.05 vs. control (0).

PC2 Enzyme Activity

We provided evidence that two PC enzymes, PC1 and PC2, were present in RIN cells. In addition, PC1 expression was altered by CPF. Decreased insulin levels can be caused by decreased activities of PC enzymes, which convert proinsulin to insulin.

PC1/3 was not detected due to the lack of a specific inhibitor. To test whether the activity of PC2 was affected by CPF, we analyzed PC2 activity using a synthetic peptide substrate in the presence or absence of an inhibitor to PC2. At 0.1 and 5 µM

CPF, the slopes of the PC2 activity were not significantly different from control. On the contrary, at concentrations from 10200 µM CPF, there was a significant decrease in the slopes, indicating inhibition of the PC2 enzyme activity (Figure 28).

To confirm that the peptide substrate and CT peptide inhibitor were present, we analyzed the reaction mixture by mass spectrometry. A peak of 828 Dalton was observed, characteristic of the substrate and a unique peak at 689 Dalton representative of the cleaved portion of the substrate. A peak at 1773 Dalton was observed in the

“inhibited” samples indicating the CT peptide without the paired basic residues at the

Cterminals, i.e. KK. A separate spectrum showed that CT peptide peaks at 2029 Dalton indicating its original peptide mass before the cell lysate was added (Figure 29).

** **

FIG. 28. PC2 enzyme activity determination after 4 hr exposure to CPF. RIN cells were treated with 0 (0.1% acetonitrile as control), 0.1, 5, 10, 25, 50, 100, and 200 µM CPF for 4 hr.

Cells were then lysed and proteins were extracted. Total proteins (30 µg) of each sample were incubated with PC2 reaction buffer in the presence and absence of CT peptide. The activity inhibited by the presence of CT peptide was considered as a PC2specific activity.

PC2 enzyme activity was expressed as fluorescence intensity. X Axis represents minutes after CPF treatment. Y Axis represents fluorescence activity. The values are expressed as the mean ±SEM. Data represents three independent experiments. Comparisons of multiple regression lines were performed using Regression Analysis. Contrast was used for Posthoc comparison. ** Donates p<0.01 vs. control.

FIG. 29. Mass spectrometry analysis on PC2 enzyme activity and its inhibitor, i.e., CT peptide. The upper spectrum indicated the PC2 activity in the presence of the substrate

(extracted from RIN cells treated with 200 µM for 4 hr). Molecular weight 828 Dalton represents the substrate and 689 Dalton represents the cleaved substrate; the middle spectrum indicated PC2 activity in the presence of the substrate and the CT peptide.

Molecular weight 1773 Dalton represents cleaved CT peptide; the lower spectrum indicated in the presence of the substrate and the CT peptide only.

Discussion

ChlorpyrifosInduced Cell Death

CPF is an organophosphate pesticide that is used worldwide in agricultural and nonagricultural applications. Thus, it is very important to continuous to evaluate its toxicity. While effects on cholinesterase have been extensively studied, much less is known about its potential toxic effects on the pancreas, especially pancreatic βcells.

The goal of the first aim was to determine whether CPF could induce apoptosis in pancreatic βcells and secondly to try to determine the underlying mechanism(s) for apoptosis. Our results suggest that CPF has both apoptotic and necrotic effects on RIN cells. Other studies have shown that cytotoxicity via apoptosis and necrosis usually appears at much higher doses, i.e. in the micromolar range (Nakadai et al. 2006;

Caughlan et al. 2004; Mense et al. 2006; Saulsbury et al. 2008). In contrast, the concentration to inhibit AChE inhibition by CPF/CPO is within the nanomolar range.

This suggests that apoptosis and necrosis are not induced by AChE inhibition, but through different mechanisms. In comparison to other cell lines (Table 1), the doses of

CPF used to cause RIN cell death are in the micromolar range.

It is commonly accepted that toxicity of CPF involves the formation of CPO, a potent anticholinesterase inhibitor. However, some studies suggest that CPF is more toxic than CPO (Nakadai et al. 2006). At 143 µM and 285 µM, CPF caused 5 times more cell death than CPO in a human monocyte cell line U937 (Nakadai et al. 2006).

Caughlan et al. also found that CPO was only slightly more potent than CPF in inducing cortical neuron apoptosis (Caughlan et al. 2004). The finding that CPF has the ability to

75 inhibit axonal outgrowth in the presence of the CYP450 inhibitor SKF525A (Howard et al. 2005) suggested that not only CPO, but also CPF injures neurons. A study by

Saulsbury et al. suggested that CPFcaused reduction in JAR cell viability was higher than that of CPO (Saulsbury et al. 2008). The studies described above demonstrate that specific cells have different sensitivities to CPF and CPO, and the cytotoxicity caused by CPF cannot be solely explained by its activated metabolite, CPO. However, potential correlations between AChE inhibition and CPFinduced apoptosis needs further study. The mechanisms by which CPF causes apoptosis and necrosis are unclear.

The proportion of apoptosis to necrosis suggests that the apoptotic pathway is predominant except at its lowest concentration (50 µM) and shorter time (2 hr) where the percentage of cell death by apoptosis and necrosis was equal.

Oxidative Stress induction by CPF

Various studies have indicated that OPs may cause oxidative stress in vivo and in vitro , as well as in humans (Ahmed et al. 2000; Akhgari et al. 2003; Bebe et al. 2003;

Sharma et al. 2005; Giordano et al. 2007). Oxidative stress generated by CPF possibly is through disruption and/or depletion of endogenous antioxidants, formation of reactive oxygen species, increased levels of oxidized glutathione (Giordano et al. 2007) and alteration of gene expression corresponding to antioxidants, such as superoxide dismutase and glutathione peroxidase (Slotkin et al. 2007). Previous studies have suggested that antioxidants could protect against the oxidative stress induced by OPs

(Gultekin et al. 2001; Gupta et al. 2001; Karaoz et al. 2002; Oncu et al. 2002;

Cankayali et al. 2005). In our study, the antioxidant, NAC, showed a small but

76 significant inhibition of CPFinduced apoptotic cell death. Necrosis was not inhibited by NAC. These results suggest a correlation between CPF and oxidative stressrelated apoptosis.

Involvement of the JNK and p38 Pathway in CPFInduced Cell Death

Transcription factors such as AP1, p53 (Minden et al. 1997; Davis 2000) and nontranscription factors such as Bcl2 family members are closely related to apoptotic cell death (Lin 2003). Various environmental stresses, such as oxidative, UV radiation, heat shock, chemotherapeutic agents, and proinflammatory cytokines are able to activate JNK. Substantial evidence indicates that JNK is an important proapoptotic factor in oxidatively stressed cells (Aoki et al. 2002; Kharbanda et al. 2000; Yamamoto et al. 1999; Lei et al. 2003; Putcha et al. 2003). While JNK is readily activated by ROS; blocking JNK activation provides significant protection against apoptosis. Activated

JNKinduced apoptosis begins in mitochondria. Bcl2 family members are believed to link JNK and apoptosis in mitochondrial apoptosis. Active JNK induces cytochrome C release from isolated mitochondria in a cellfree assay (Aoki et al. 2002) and when cells are in stress, JNK translocates to the mitochondria (Kharbanda et al. 2000). Evidence suggests that JNK induces apoptosis by regulating Bcl2 family members. For example,

JNK phosphorylation inhibits the antiapoptotic function of Bcl2 (Yamamoto et al.

1999) and enhances the proapoptotic Bcl2 family member, Bim through phophorylation (Lei et al. 2003; Putcha et al. 2003) along with conformational changes and translocation of Bax (Lei et al. 2002; Tsuruta et al. 2004). Our data demonstrates that the JNK inhibitor, SP600125 (20 µM) almost completely blocked CPFinduced

77 apoptosis. This suggests that the JNK pathway is activated by CPF. As the inhibition of oxidative stress by NAC was not able to completely block apoptosis, it would appear that other factors besides oxidative stress are involved in JNK activation.

Oxidative stress is believed to have the ability to induce either apoptosis or necrosis. However, why and how cells die from necrosis is far from clear. Apoptosis and necrosis may or may not occur at the same time and at the same exposure dose.

Mense et al. (Mense et al. 2006) found that CPF caused LDH release in primary human astrocytes at a concentration as low as 0.2 µM after 2 weeks treatment, whereas at 100

µM, CPF generated apoptosis as analyzed by TUNEL. However, the mode of cell death is dependent on the cell type, the extent of oxidative stress, and the cell signaling pathway elicited by ROS (Gardner et al. 1997; Troyano et al. 2003). In RIN cells, ROS does not mediate CPFinduced necrotic cell death since the antioxidant NAC did not inhibit RIN cell necrosis. However the result that the JNK inhibitor, SP600125, partially blocks CPFinduced necrosis suggests that JNK may be involved in RIN necrosis. Further work is needed to identify the molecular targets of JNK in

ROSinduced apoptotic and necrotic cell death.

P38 is also a MAPK family molecule. Along with JNK, it is preferentially activated by cell stressinducing signals, such as oxidative stress and toxic chemicals

(Davis 2000). P38 inhibition failed to attenuate CPFinduced placental cell apoptosis

(Saulsbury et al. 2008). Similarly, in rat cortical neurons, blocking p38 significantly accelerated apoptotic cell death in the presence of CPF (Caughlan et al. 2004). In our experiments, the p38 inhibitor, SB202190, partially blocked CPFinduced cell

78 apoptosis suggesting that p38 acts via a proapoptotic pathway to mediate CPFinduced

RIN apoptosis. This implies that there are no absolute signaling pathways and the role of activated p38 varies in different cells. Currently, there are very limited resources in regard to the activation of p38 and the following molecular action of apoptosis. P38 induced apoptosis was reported to regulate Bax translocation (Shou et al. 2003). Bax translocates from the cytosol to the mitochondria to disrupt mitochondrial membrane potential and to heterodimerize with antiapoptotic Bcl2 proteins to counteract their actions. Similar to the mechanism in JNKinduced apoptosis, p38 was also found to directly phosphorylate Bim at Ser65 in sodium arseniteinduced apoptosis (Cai et al.

2006). Our results suggest that necrotic cell death was not prevented by SB202190, an inhibitor of P38. Thus, we would conclude that the P38/MAPK pathway is not contributing to necrosis in RIN cells treated with CPF.

Effect of Chlorpyrifos on Insulin Processing and Secretion

Previous data from our laboratory showed that cutaneous CPF exposure led to insulin reduction in the circulation and decreased insulin content in rat pancreas

(Rutherford and Cool, unpublished data). This specific aim is to investigate whether insulin secretion is impaired at lower concentrations than lethal doses and its potential mechanism(s).

This is the first study demonstrating the effects of CPF on insulin secretion in pancreatic βcells. Two secretagogues, potassium and glucose were used to evaluate insulin secretion. The metabolic signaling pathways of glucose and potassium leading to insulin secretion are different (Figure 30). Glucose has been the most intensively studied secretagogue. The rise in plasma glucose levels results in an increased flux of glucose into pancreatic βcells through the glucose transporter 2 (GLUT2) (Thorens

1992). The cytosolic glucose is then metabolized to glucose6phosphate by glucokinase, the ratelimiting step in glycolysis (De Vos et al. 1995; Newgard et al.

1995). The result of glycolysis is the production of ATP to increase the cytosolic

ATP/ADP ratio (Reimann et al. 2000). In the pancreatic β cells, an increased ATP/ADP

+ ratio will close the ATPsensitive K (K ATP ) channels and result in depolarization that in turn opens voltagedependent Ca 2+ channels (VDCCs) resulting in Ca 2+ influx. Insulin secretion is directly triggered by this Ca 2+ influx.

The K ATP channel is a heterooctamer composed of 4 units, Kir6.2 with 4 sulfonylurea receptors (SUR1) (Miki et al. 1999). Sulfonylurea drugs for diabetes are agonist (ligands) of SUR and can thereby close the K ATP channels independent of

80 metabolic activity. The mitochondria are crucial for the coupling of glucose metabolism to insulin secretion. Mutations in the mitochondria genome can cause diabetes and poor release of insulin in response to glucose (Soejima et al. 1996). Cellular models with

DNAdeficient mitochondria are mitochondrial defective and glucose unresponsive as well. However, the cell retains depolarizationmediated insulin secretion, i.e., potassium and sulfonylurea stimulated insulin secretion (Soejima et al. 1996; Kennedy et al. 1998; Tsuruzoe et al. 1998). Therefore, glucose and potassium usually are used in insulin secretion analyses to evaluate mitochondrial function. If glucosestimulated insulin secretion is abnormal and potassiumstimulated insulin is not affected, the mitochondria are usually the target. Our results showed that both potassium and glucose stimulated insulin secretion were affected by CPF. This suggests that CPF acts upstream of glucose metabolism i.e. impaired insulin synthesis, proinsulin processing and downstream of calcium influx, i.e. exocytosis process.

Our results showed that CPF did not impact basal insulin secretion. RIN cells stimulated by 50 mM potassium and 25 mM glucose showed significant stimulation in insulin secretion compared to basal. Our results support that insulin secretion in RIN cells is through regulated secretory pathway (Bhathena et al. 1984; Santangelo et al.

2007; Mao et al. 2009). However, in the presence of CPF, RIN cells exhibited a significant reduction in insulin secretion compared to control. This could be explained by changes in the processing pathway, or by changes in the membrane dynamics that may prevent fusion of the secretory granules with the membranes or with a weakened depolarization of the membrane and leakiness that would cause loss of calcium or other

FIG.30 . Scheme of steps involved in potassium and glucose stimulated insulin secretion.

1. Glucose transport in to cells through glucose transporter 2 (GLUT2)

2. Glucose metabolism in mitochondria to produce ATP

3. ATP sensitive potassium channel close s

4. Open of voltage dependent Ca 2+ channels (VDCCs)

82 required ions necessary for secretion. Proinsulin, however, produced by RIN cells at basal level was higher than stimulated status (in the presence of 50 mM potassium and 25 mM glucose). While many studies have shown that proinsulin processing in healthy individuals is in regulated secretory granules (Halban 1991; Halban 1994), analysis in mouse β TC3 insulinoma cells concluded that newly synthesized proinsulin was released predominantly via a constitutive, rather than a regulated pathway (Nagamatsu et al.

1992). In addition, studies in mouse pituitary AtT20 cells also supported that in the process of prohormone proopiomelanocortin (POMC), a large fraction of incompletely processed POMC was secreted from immature secretory granules instead of mature granules in a constitutivelike pattern (Dumermuth et al. 1998).

Based on our results and other studies, we propose that in RIN cells, proinsulin and their intermediates are sorted out of the immature granules to constitutivelike vesicles and secreted constitutively (Figure 31B).

CPF concentrations used in our studies were 5200 µM. Based on pharmacokinetic models; Buratti et al. proposed that OP concentrations higher than 100 µM reflect acute accidental intoxication (Buratti et al. 2007). Currently, we do not know how long

CPFinduced decreased insulin secretion will last and whether it is applicable in humans. Providing CPF induces decreased insulin secretion in humans, the situation will be worse for those who have diabetes. RIN cells treated with CPF showed a trend toward decreased intracellular insulin after 4 hr stimulation with 50 mM KCl and 25 mM glucose. Inhibition of insulin biogenesis, i.e. inhibition of proinsulin synthesis is probably not the explanation as intracellular proinsulin did not change accordingly. In 83

(A)

(B)

FIG.31. Scheme of proinsulin processing in normal β cells via regulated and RIN cells via constitutive secret ory pathway. RER, Rough Endoplamic Reticulum; TGN,

TransGolgi Network.

order to maintain normal insulin secretion and its content in βcells, the balance between proinsulin and insulin is very important. Prohormone convertase (PC) enzymes are responsible for converting proinsulin to insulin. Any deviation of PC enzymes will cause compromised insulin secretion. The deviation includes the changes in PC enzyme expressions and their activities. Our results indicated that active PC1 in

RIN cells treated with CPF was upregulated at 4 hr postexposure. CPF produced no change on PC2 expression by western blotting. The PC2 enzyme activity assay for the first time showed that PC2 was inhibited by CPF. An analysis of the slopes of the activity assay in which a synthetic substrate was cleaved by PC2 showed that the slopes of the lines for the chlorpyrifos treated cells decreased with increasing concentrations of chlorpyrifos. The importance of the decreased slope is that CPF is inhibiting the PC2 enzyme activity. Inhibition of PC2 activity would result in decreased processing of proinsulin to insulin. The buildup of proinsulin in the cell would trigger mechanisms for removal of the extra proinsulin from the regulated secretory granules to the constitutivelike secretory granules/pathway. These results suggest a mechanism for decreased insulin secretion and cellular content. Further experiments are needed to ascertain that (i) PC1 enzyme activity is affected by CPF; (ii) PC enzyme activity inhibition is via CPF phosphorylation at an active serine site.

The other possible candidates that could be responsible for impaired insulin secretion and decreased intracellular content could be (i) inhibition of proinsulin conversion to insulin other than inhibited PC2 activity, (ii) interrupted insulin

85 exocytotic process and (iii) accelerated insulin degradation (Halban et al. 1980; Rhodes et al. 1988).

A selective inhibitor peptide (CT peptide) was used that was purported to bind with high affinity to the PC2 enzyme active site, in the experiments on PC2 enzyme activity.

When the reaction buffer containing all the components of the assay was analyzed using mass spectrometry, the paired lysine residues at the Cterminus of the CT peptide were found to be removed. Theoretically this should only happen by cleavage with carboxypeptidases, as are found in the secretory granules of insulin secreting cells.

However, since other enzymes are present in the lysates used for the assay, other enzymes may also play a role in removal of the lysine residues. The significance for this observation is that the pairedbasic residues are the consensus signal sequence for cleavage by PC1 and PC2. Their removal may cause the peptide to be removed from the active site or it may make it bind tighter. No one has yet described this reaction using mass spectrometry. Therefore, it is unknown what is happening at the enzymesubstrate level.

Conclusions

Poisoning by organophosphate insecticides is a major health concern. In addition to cholinergic effects, hyperglycemia has been observed in both acute and chronic exposure to OPs. Different mechanisms have been studied for OPinduced hyperglycemia. However, little is known regarding the effects on pancreatic βcells. We used CPF to study the toxicity on insulinsecreting βcell line RINm5f. Our results showed that CPF induces a sharp decline of cell survival at concentration of 50 µM as early as 2 hr treatment. CPF not only induces apoptosis but also necrosis in RIN cells.

The later mechanistic studies indicated that apoptotic death in RIN cells are through

JNK and p38 pathways. ROS is also involved in CPFinduced apoptosis. However,

CPFinduced necrosis has different pathways. Four hour exposure of RIN cells to CPF concentrations lower than lethal doses (5, 10 and 25 µM) significantly impaired potassium and glucosestimulated insulin secretion. Our results imply that for at least a transit time, CPF induces a reduction in insulin secretion and cellular insulin which potentially leads to hyperglycemia and diabetic conditions. Decreased insulin secretion and insulin content can be explained by inhibition of PC2 enzyme activity with no change in the enzyme protein. In addition, our data, for the first time showed that proinsulin secretion in RIN cell is through constitutive pathway instead of regulated pathway. In conclusion, our observation shows that pancreatic βcells are specific and sensitive targets for CPF toxicity. Decreased insulin secretion by CPF can be explained by reduction in βcell viability and inhibition of PC enzymes.

Future Study

Molecular Targets of JNK and p38

Currently, JNK and p38 have been shown to medicate two distinct apoptotic pathways (Aoki et al. 2002; Davis 2000; Dhanasekaran et al. 2008; Kharbanda et al.

2000; Lei et al. 2003; Lin 2003; Minden et al. 1997; Tsuruta et al. 2004). One is called a mitochondrial pathway; the other is a nuclear pathway. In the mitochondrial pathway, activated JNK or p38 interacts with antiapoptotic proteins, such as Bcl2, and proapoptotic proteins, such as Bim and Bax to induce cytochrome C release from mitochondria leading to activation of caspase 3 dependent apoptosis. In the nuclear pathway, activated JNK or p38 can also translocate to nucleus to phosphorylate cJun and/or p53 to initiate transcription of proapoptotic gene such as Bax to induce apoptosis.

Our data suggest that JNK and/or p38 mediate(s) CPFinduced RIN cell apoptosis.

However, the involvement of downstream molecular targets such as Bcl2, Bax, cJun and p53 needs further study.

PC1 Activity Detection

According to our hypothesis, both PC1 and PC2 enzyme activities should be inhibited by CPF. However, PC1 enzyme activity was not able to be detected because of the lack of a specific PC1 inhibitor. A new method needs to be developed to analyze

PC1 enzyme activity.

Prohormone Convertase Catalytic Domain Serine Phosphorylation by CPF

To test if the inhibition of PC enzyme activity is through phosphorylating the serine site at their catalytic domains, mass spectrometry is needed to be carried out to detected

88 phosphorylation modification on PC enzyme.

Insulin Secretion Changes in Primary βcell line and In Vivo Study

Since RIN cells are rat insulinoma cells, studies on primary βcell line and in vivo studies are necessary to expand our conclusions. The results from these in vivo studies would provide the framework for a translational approach to studying the effects of

CPF on humans.

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