o paraoxon neithers diabetes mellitus

Mohamed Shaullah,a Huba Kalsz,c

ruunxtredlinexd kisy hicand

early 1800s by Moschnine (Petroianu, 2008). The process was Other sources of exposure include food chain, contaminated drink-

Research Article

ted: 2 June 2012 Published online in Wiley Online Library: 9 August 2012

1036rst published in 1854 by de Clermont (Kenneth et al., 2008).OPCs were primarily synthesized for crop protection againstinsect pests but were later used to produce deadly poison suchas nerve agents. OPCs are classied into many groups which arestructurally and toxicologically different. However, a commonmechanism of action is found in all of the different groups ofOPCs: the irreversible inhibition of acetylcholinesterase (EC 3.1.1.7)with the active centre being the serine hydroxyl group (Delnoet al., 2009). Inhibition of acetylcholinesterase results in theaccumulation of acetylcholine at nerve endings leading to over-stimulation of neurons. Each OPC has a unique structural prolefor toxicity and behavior, ranging from extremely toxic nerveagents to a moderate or slightly toxic OPCs used in pesticides. It

ing water and air. OPCs accounts for several hundreds of thousandsof deaths worldwide every year (Karalliedde and Senanayake, 1999).

*Correspondence to: E. Adeghate, Department of Anatomy, Faculty of Medicine& Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates.E-mail: eadeghate@uaeu.ac.ae

aDepartment of Pharmacology and Therapeutics, Faculty of Medicine andHealth Sciences, United Arab Emirates University, UAE

bDepartment of Cellular Biology & Pharmacology Herbert Wertheim College ofMedicine, Florida International University, USA

cnor in POX-treated normal rats. Electron microscopy of POX-treated pancreas did not show any morphological changesin beta cells. These results suggest that POX does not cause diabetes mellitus at sub-lethal sub-chronic exposure.Copyright 2012 John Wiley & Sons, Ltd.

Keywords: paraoxon; diabetes; acetylcholinesterase; hyperglycemia; sub-lethal dose; diabetogenic

IntroductionOrganophosphorus anticholinesterase compounds are esters,amides or thiol derivatives of phosphoric, phosphonic, phosphi-nic acids, and phosphorothioic or phosphonothioic acids. Thephosphonic acids derivatives are more toxic than the phospho-ric acids, whose oxygen atom can be substituted by sulphur ornitrogen atoms (Bosak, 2006). The rst synthesized organophos-phorus compound (OPCs) was a monoester, named tetraethylpyrophosphate (TEPP). The compound was synthesized in the

including neuropathy, pancreatitis, metabolic disruption andreproductive impairment (Slotkin, 2011; Washam, 2008).

OPCs are one of the most widely used pesticides worldwide.According to a previous report, 50% of all insecticides used in theworld belong to the OPC group (Casida and Quistad, 2004). Thesepesticides are a source of occupational hazards, especially for farm-ers and pesticides applicators. Its domestic exposure is also commonbecause of the use of insecticides for roaches, ies, ticks, mites andother insects. The unintentional or accidental exposure as well assuicidal use is very common, particularly in third world countries.Sub-chronic exposure tinduces nor exacerbatein Wistar ratSyed M. Nurulain,a Georg Petroianu,b

Murat Oz,a Tariq Saeed,d Abdu Adem

ABSTRACT: There is an increasing belief that organophosphohyperglycemia and diabetes mellitus. The present study wassub-lethal and sub-chronic exposure to paraoxon (POX), an eoxon on streptozotocin-induced diabetic rats was also examinfor 6 weeks. Blood glucose levels and red blood cell acetylchoand morphological studies were performed at the end of theexacerbates diabetes mellitus in experimental rats. Liver andifferences when compared with controls. Biochemical analysin protein level in all groups. Urine bilirubin was signicantlcontrol group. The number of blood cells in urine was signcontrol group. Hyperglycemia was noted in the diabetes

Received: 23 February 2012, Revised: 1 June 2012, Accep

(wileyonlinelibrary.com) DOI 10.1002/jat.2794is well known that heavy exposure to OPC causes acute cholinergicsyndromes, which may lead to high mortality and morbidity.Moreover, chronic or sub-chronic exposure to low doses ofOPC over a period of time may cause a variety of health problems

J. Appl. Toxicol. 2013; 33: 10361043 Copyright 2012 Johna and Ernest Adeghated*

s compounds (OPCs) impair glucose homeostasis and causedertaken to investigate the putative diabetogenic effect ofemely hazardous OPC used in pesticides. The effect of para-. Each rat was injected with 100 nmol of POX 5 days per weekesterase activity were measured weekly. Biochemical analysisperiment. The results revealed that POX neither induces noridney/body weight ratios revealed statistically insignicantof urine samples showed a small but not signicant increaseigher in the diabetes + POX group when compared with theantly higher in the POX-treated group compared with thediabetes + POX groups, but neither in the saline controlDepartment of Pharmacology and Pharmacotherapy, Semmelweis University,Budapest, Hungary

dDepartment of Anatomy, Faculty of Medicine & Health Sciences, United ArabEmirates University, Al Ain, United Arab Emirates

Wiley & Sons, Ltd.

The exact mechanism responsible for the OPC-induced type 2diabetes is unclear but the proposed mechanisms include oxida-

Group 1 (G1): Saline control

Germany) as described previously (Worek et al., 1999). The

acetylthiocholine. The change in the absorbance of DTNB

Paraoxon and diabetes mellitus

10tive stress, nitrosative stress, inhibition of paraoxonase, inductionof inammatory cytokines, stimulation of the adrenal gland, dis-turbed metabolism of the liver and inhibition of cholinesterases(Rahimi and Abdollahi, 2007; Li and JianShe, 2009; Everettand Matheson, 2011) and disturbance in glucose homeostasis(Abdollahi et al., 2004; Rezg et al., 2010; Kamath and Rajini, 2007).The majority of investigators reported that exposure to OPCinduces hyperglycemia (Ambali et al., 2011; Rahimi and Abdollahi,2007; Seifert, 2001). According to Abdollahi et al. (2004) andSlotkin (2011) OPCs are predisposing factor of diabetes mellitus.

Type 2 diabetes is characterized by insulin resistance, which iscompensated initially by increased insulin production. Over thecourse of the disease, the pancreas fails to produce sufcientinsulin to stimulate adequate glucose uptake in adipose andmuscle tissues leading to hyperglycemia and type 2 diabetes.Animal studies have demonstrated that tolerance to OPCs expo-sure develops over time, most probably as a result of decreasedexpression of muscarinic receptors (Costa et al., 1982). This maypotentially lead to a decrease in insulin production. Moreover,prolonged acetylcholine release may reduce cells sensitivityto glucose (Gilon and Henquin, 2001).

The objective of this study was to investigate the possiblediabetogenic effect of a sub-lethal dose and sub-chronic exposureto paraoxon (POX), an OPC compound, in adult male Wistar rats.

With the exception of azinfos-methyl, the majority of the testedcompounds belong to class III i.e. slightly hazardous, according toWHO classication of pesticides (World Health Organisation,2009). Therefore, it is important to determine whether POX, anextremely hazardous OPC, is diabetogenic or not.

Material and Methods

Experimental Animals

The original stock of Wistar rats was purchased from HarlanLaboratories (Harlan Laboratories, Oxon, England). The animalsused in the actual experiments were bred at our own AnimalFacility from the original stock. Male adult rats, weighingbetween 200 and 250 g were used in this study. The animalswere housed in polypropylene cages (43 22.5 20.5 cm; sixrats per cage) in climate and access controlled rooms (23 1 C;50 4% humidity). The day/night cycle was 12/12 h. Food andwater were available ad libitum. The food was a standard mainte-nance diet for rats and was purchased from Emirates Feed Factory(Abu Dhabi, UAE). All the animals procedures were carried outAlthough, the main target of OPC action is the central andperipheral nervous systems, there is growing evidence thatOPC may cause a lot more disorders outside of the central andperipheral nervous systems (Lukaszewicz-Hussain, 2010; Washam,2008). For example, clinical and animal model studies have shownthat some OPCs cause hyperglycemia after acute exposure(Husain and Ansari, 1988; Matin and Hussain, 1987; Meller et al.,1981; Shobha and Prakash, 2000). A summary of studies thatshowed a link between OPCs and hyperglycemia is given inTable 1AD. According to Saldana et al. (2007) and Rezg et al.(2010) there is a link between insecticide exposure and diabetesmellitus. Montgomery et al. (2008) identied 10 OPC insecticidesthat cause diabetes in humans, especially after long-term expo-sure. These agents include chlorpyrifos, diazinon and trichlorfon.J. Appl. Toxicol. 2013; 33: 10361043 Copyright 2012 Johnwas measured at 436 nm. The RBC-AChE activity was calcu-lated using an absorption coefcient of TNB at 436 nm(e= 10.6 mM1 cm1). All enzyme activities were expressed asfreshly drawn venous blood was diluted 1:100 in a solution(0.1 M phosphate buffer + Triton-X) and immediately frozen(Worek et al., 1999). The enzyme activity was measured usinga Milton Roy Spectronics 301 spectrophotometer (Milton Roy,Ivyland, PA, USA). Samples were kept at 20 C until analysis.The assay, which is based on Ellmans method, measures thereduction of dithiobis-nitrobenzoic acid (DTNB) to nitrobenzoate(TNB-) by thiocholine, the product of acetylthiocholine hydrolysis(Ellman et al., 1961). The samples were diluted in 0.1 M phosphatebuffer (pH 7.4) and incubated with DTNB (10 mM) and etho-propazine (6 mM) for 20 min at 37 C prior to the addition ofChemicals

POX stock solution (100 mM) was prepared in dry acetone. Theworking solution for i.p. application was prepared ex temporeby diluting the stock solution with saline. Paraoxon-ethyl andstreptozotocin were purchased from Sigma-Aldrich Chemie(Sigma-Aldrich Chemie GmbH, Steinheim, Germany).

Red Blood Cell-Acetyl-Cholinesterase Activity

The blood samples for red blood cell-acetyl-cholinesterase(RBC-AChE) measurement were collected from the tail vein.The RBC-AChE activity was measured in diluted whole bloodsamples in the presence of the selective butyryl-cholinesteraseinhibitor, ethoproprazine (Sigma-Aldrich Chemie, Steinheim,Group 2 (G2): POX only treated group.Group 3 (G3): Diabetes control group (No treatment with POX)Group 4 (G4): Diabetes group treated with 100 nmol per ratof POX.

The animals were treated daily for 5 days followed by a 2-daybreak in a 7-day cycle, for 6 weeks.with strict compliance to the Ethical Committee for the care anduse of laboratory animals.

Model of Diabetes Mellitus

After an overnight fast, rats for the model of type 1 diabeteswere rendered diabetic by a single intra-peritoneal (i.p.) injectionof streptozotocin (60 mg kg1 body weight) in a freshly preparedcitrate buffer (0.1 M pH 4.5) solution. The streptozotocin-injectedanimals exhibited hyperglycemia within 24 h. Diabetes instreptozotocin-treated rats was conrmed using a One-touchGlucometer (Lifescan Inc., Milpitas, CA, USA). Animals with bloodglucose above 235 mg dl1 were considered diabetic and wereused for the experiments.

Experimental Design

There were four groups of 610 rats. POX was given i.p. at adose of 100 nmol per rat. The dose of POX was the maximumtolerable for chronic study. It was used by Hasan et al. (2004)in a 6-week treatment protocol.Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jat

37

Table 1. A. Studies on rats that show the effect of organophosphorus compounds (OPCs) onbloodglucose homeostasis in acute application

Study Type of OPC Reported result on glycemia

Kuzminskaia et al., 1978 Valexon (Class III: slightly hazardous) Disturbance in glucose homeostasisRodrigues et al., 1986 Malathion (Class III: slightly hazardous) Transient hyperglycemiaMatin and Hussain, 1987 Malathion (Class III: slightly hazardous) HyperglycemiaHusain and Ansari, 1988 Diazinon Class II: Moderately hazardous HyperglycemiaMatin et al., 1990 Diazinon (Class II: Moderately hazardous) HyperglycemiaShih and Scremin, 1992 Soman (Deadly poison nerve agent) Increased blood glucoseIkizceli et al., 2005 Fenthion Class II: Moderately hazardous) HyperglycemiaPanahi et al., 2006 Malathion (Class III: slightly hazardous) Increased blood glucoseRomero-Navarro et al., 2006 Dichlorvos (Class IB: Highly hazardous) No effect on glucose metabolism.Lasram et al., 2008 Malathion (Class III: slightly hazardous) Transient hyperglycemiaJoshi and Rajini, 2009 Acephate (Class III: slightly hazardous) Reversible hyperglycemiaKrishna and Ramachandran, 2009 Chlorpyriphos (Class II: Moderately hazardous) Co-administration of Chlorpyrifos

and lead causes hyperglycemiaJoshi and Rajini, 2012 Monocrotophos (Class IB: Highly hazardous) Reversible hyperglycemiaRuckmani et al., 2011 Malathion (Class III: slightly hazardous) Transient hyperglycemia

B. Studies on rats that show the effect of organophosphorus compounds (OPCs) on blood glucose homeostasis in chronic/sub-chronic application

Study Type of OPC Reported result on glycemiaGupta, 1974 Malathion ( Class III: slightly hazardous) No effectDeotare and Chakrabarti, 1981 Acephate (Class III: slightly hazardous) Increased blood glucoseReena et al., 1989 Dimethoate (Class II: Moderately hazardous) HyperglycemiaSarin and Gill, 1999 Dichlorvos (Class IB: Highly hazardous) HyperglycemiaSeifert, 2001 Diazinon (Class II: Moderately hazardous) HyperglycemiaHagar and Fahmy, 2002 Dimethoate (Class II: Moderately hazardous) Increased blood glucoseAbdollahi et al., 2004 Malathion (Class III: slightly hazardous) HyperglycemiaPournourmohammadi et al., 2005 Malathion (Class III: slightly hazardous) HyperglycemiaRezg et al., 2006 Malathion (Class III: slightly hazardous) No change in blood glucosePanahi et al., 2006 Malathion (Class III: slightly hazardous) HyperglycemiaPournourmohammadi et al., 2007 Malathion (Class III: slightly hazardous) HyperglycemiaKamath and Rajini, 2007 Dimethoate (ClassII: Moderately hazardous) Increased blood glucoseSadeghi-Hashjin et al., 2008 Azinfos methyl, Malathion (Class IB , III) No diabetogenic effectAmbali et al., 2011 Chlorpyrifos (ClassII: Moderately hazardous) HyperglycemiaWang et al., 2009 Chlorpyrifos (Moderately hazardous) Increased blood glucoseSlotkin, 2011 Chlorpyrifos (Moderately hazardous) Normal serum glucoseBegum and Rajini, 2011 Monocrotophos (Class IB: Highly hazardous) Marginal increaseRuckmani et al., 2011 Malathion (Class III: slightly hazardous) Progressive hyperglycemia

C. Clinical studies/case reports that show organophosphorus compounds (OPCs)-induced hyperglycemia in patients.

Study Type of OPC Reported result on glycemiaMartn et al., 1996 OP compounds Pancreatitis and central diabetes insipedusMeller et al., 1981 OP poisoning Diabetes mellitus and non ketotic

hyperglycemiaMoore and James, 1981 OP poisoning Hyperglycemia, glycosuria, ketonuriaShobha and Prakash, 2000 OP poisoning Transient glycosuria in patients.Wu et al., 2001 methamidophos HyperglycemiaYanagisawa et al., 2006 Sarin (Deadly toxic/nerve agent) Hyperglycemia, ketonuriaAkyildiz et al., 2009 OP poisoning Diabetic ketoacidosis.Kumar and Nayak, 2011 OP poisoning Diabetic ketoacidosis.

D. Epidemiological studies that show the risk of developing type 2 diabetes after chronic exposure to organophosphoruscompounds (OPC).

Study Type of OPC Reported result on glycemiaSaldana et al., 2007 Diazinon (Class II: Moderately hazardous) Gestational diabetes mellitusMontgomery et al., 2008 Dichlorvos (Class IB: Highly hazardous) Risk of diabetes increase with exposure days

S. M. Nurulain et al.

J. Appl. Toxicol. 2013; 33: 10361043Copyright 2012 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/jat

1038

solution (Karnovsky, 1965). The glands were washed overnight

POX-treated rats group was signicantly lower (Bonferroni factor6) compared with the saline control (Fig. 1). Intraperitoneal injec-

Figure 1. Mean red blood cell-acetyl-cholinesterase (RBC-AChE) activity6 weeks after treatment. Note a signicant (P< 0.05) reduction in activityafter paraoxon (Pox) treatment and diabetes with Pox treatment. Dataare mean standard deviation (SD). *Shows the statistical signicancewhen compared with the corresponding control.

Figure 2. Body weight in percentage, from 06 weeks of treatment. Notethat Pox prevented the loss of body weight in the streptozotocin-induceddiabetic rats.

Paraoxon and diabetes mellitus

10in cacodylate buffer (4C) and post xed with 1% osmium tetrox-ide for 1 h. The samples were later washed six times in cacody-late buffer and dehydrated in graded concentrations of ethylalcohol. They were later treated with propylene oxide; twochanges of 15 min and immersed in propylene oxide; and resin(1:1) for 1 h followed by pure resin overnight. The samples wereembedded and polymerized at 60 C for 24 h. Semi- and ultra-thin sections were cut using a diatome knife (supplied by AgarScientic, Essex, England). The semithin sections were placedon glass slides and stained with toluidine blue while the ultra-thin sections were mounted on 3.0 mm 200 mesh copper gridsand contrast stained with saturated aqueous uranyl acetatefor 30 min and Reynolds (1963) lead citrate for 5 min. The ultra-thin sections were viewed with a Philips CM10 transmissionelectron microscope (Eindhoven, The Netherlands).

Statistical Analysis

Statistical analysis was performed on all the experimental para-meters using SPSS 11.0 (SPSS Inc.). The MannWhitney rank ordertest was used to determine the signicance in comparison to con-trol values. The limit of statistical signicance was set at P 0.05.

ResultAcetylcholinesterase activity of RBC

Acetylcholinesterase enzyme activity of RBC was determined asa percentage of baseline. The enzyme level in the RBC of theBiochemical Test of Urine

After 6 weeks of treatment, a semi-quantitative biochemical test ofurine samples was done using Combur10-Test strips (Boehringer,Mannheim, Germany). The urine was collected in sterile petri-dishand the dipstick was soaked and the color change was readaccording to the information given in the instruction manual.Statistical analysis using SPSS 11.0 was performed. The limit ofstatistical signicance was set at P 0.05.

Organ and Body Weight

At the end of 6 weeks, the rats were decapitated using a guillotineand the liver and kidney were removed, weighed and comparedwith the total body weight.

Electron Microscopy

Samples of the pancreas from all four groups were cut into smallpieces and xed for 5 h in 2.5% glutaraldehyde in Karnovskysa percentage of baseline activities (100%). The sums of enzymeactivities over time in individual animals (6 weeks) were com-pared using the MannWhitney rank order test using SPSSsoftware (SPSS Inc. Chicago, IL, USA). P 0.05 was consideredto be statistically signicant.

Blood Glucose Measurement

Blood glucose was checked with a One-touch Gucometer (LifescanInc., Milpitas, CA, USA) on day 5 of each week of a 7-day cyclebefore the application of the 5th injection of POX. The bloodsamples were collected from the tail vein.J. Appl. Toxicol. 2013; 33: 10361043 Copyright 2012 Johntion of 100 nmol POX to rats in groups G2 and G4 caused49 13 and 49 6 (mean % SD) inhibition of RBC-AChE activ-ity, respectively. Six weeks of STZ-induced diabetes (G3) resultedin a 15 5 decrease in RBC-AChE enzyme activity. The trend ofPOX-induced inhibition of RBC-AChE in G2 was progressive withno signicant difference between weekly intervals. In G4, thepercentage inhibition remained almost the same throughoutthe experimental period. In G3, there was a small but not signif-icant inhibition of RBC-AChE throughout the course of the study.

Animal body weight

Animal body weight was recorded at the end of each week(Fig. 2). The body weight of rats in group G1 rats increased sig-nicantly from week 1 to 6 in comparison with 0 week weight.In G2, the body weight increased in the same manner as thecontrol. Rats in G3 experienced a decrease in body weightthroughout the experimental period compared with the salinecontrol. Although rats in group G4 had a uctuating weight,there was no signicant increase in body weight compared withthe beginning of the experiment.Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jat

39

Glucose level

The measurement of the blood glucose level revealed no signif-icant changes in all four groups throughout the experiment(Table 2). The average blood glucose level was 63 6 mg dl1in the control and 63 7 in POX-treated group. The blood glu-cose levels for the diabetes control group (G3) and diabetic ratstreated with POX (G4) were 363 67 and 346 28 mg dl1,respectively. The MannWhitney rank order test on the datashowed no signicant differences between G1 vs. G2 andG3 vs. G4.

Organ weight/body weight ratio

To determine the effect of long-term sub-lethal exposure of POXon the liver and kidney of non-diabetic and STZ-induceddiabetic rats, the liver and kidney were weighed at the end of6 weeks and the individual organ weight was divided by

corresponding animal body weight. The ratio of kidney/bodyweight and liver/body weight was almost the same in all groups(Tables 3A and B) and no signicant differences were observed.

Urine analysis

Notable differences amongst the four groups were the presenceof bilirubin and erythrocytes in the urine samples of G2 and G4respectively (Table 4).

Electron microscopy

Figure 3 shows the electron microscopy of four groups of rats.No morphological changes were observed between the treatedand control groups. The structure of pancreatic beta and otherendocrine cells was intact, suggesting that insulin and otherhormone production was normal in the POX-treated rats (G2).The morphology of pancreatic islet cells of the diabetes onlygroup and those of diabetic rats treated with POX was similar,

Table 2. Blood glucose level (mg dl1) at weekly intervals

Groups Baseline Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Mean

Saline control (G1) 66 7 59 3 61 5 59 3 67 8 58 6 72 12 63 6Paraoxon only (G2) 71 14 56 5 66 6 54 4 70 6 60 8 71 10 63 7Diabetes only (G3) 433 50 448 74 359 52 358 45 332 106 261 102 423 93 363 67Diabetes + Paraoxon (G4) 349 48 328 67 343 61 310 72 362 79 341 87 389 42 346 28Results are expressed as mean standard deviation. The means of 6 weeks were compared with the baseline of respective groups.

Table 3. A. Liver/body weight ratio at the end of 6 weeks

Body weight (g) (Mean SD) Liver weight (g) (Mean SD) Ratio (Mean SD)Saline control 254.666 24.784 9.252 1.513 0.036 0.004

oxo

00

S. M. Nurulain et al.

1040Paraoxon treated 275.166 42.780Diabetes only 205.250 21.313Diabetes + paraoxon 221.200 32.754

B. Kidney/body weight ratio at the end of 6 weeks.Body weight (g) (Mean SD)

Saline control 254.6667 24.7844Paraoxon treated 275.1667 42.7804Diabetes only 205.2500 21.3131Diabetes + paraoxon 221.2000 32.7543

Table 4. Biochemical analysis of urine samples

Saline control Para

pH 6.00 0.00 6.

Protein (mg dl1) 13.33 12.91 17.50Glucose (mg dl1) 40 0.00 40Bilirubin (mg dl1) 0.60 0.31 2.30Blood cells (Ery ml1) < 5 0.00 90.00*The results are mean standard deviation.The detection limit of the test strip is: pH value = 59; protein (albuabove-mentioned average calculation; glucose: 401000 mg dl1. 40result is assigned a value of 0.4 mg dl1; blood cells: 5250 ery ml1

* Signicant difference compared with the saline control.** Signicant difference compared with the diabetes control.

Copyright 2012 Johnwileyonlinelibrary.com/journal/jat9.417 1.843 0.034 0.0029.665 0.984 0.047 0.047

10.042 1.243 0.046 0.005

Kidney weight (g) (Mean SD) Ratio (Mean SD)1.932 0.2543 0.0074 0.0004

1.8667 0.2703 0.0068 0.00042.3225 0.0929 0.0114 0.00082.5960 0.3617 0.0119 0.0019

n (Pox) Diabetes Diabetes + Pox

0.00 5 0.00 5.00 0.00

13.69 44.00 31.30 40.00 26.460.00 1000 0.00 1000 0.001.88 1 0.00 2.71** 1.6089.44 250.00 0.00 250.00 0.00

min): 6500 mg dl1. The negative result is assigned ve in themg dl1 is considered normal; Bilirubin: 0.56 mg dl1. A negative.

J. Appl. Toxicol. 2013; 33: 10361043Wiley & Sons, Ltd.

Paraoxon and diabetes mellitussuggesting that POX did not alter the structure of cells of theislet of Langerhans, either in normal or diabetic conditions.

DiscussionThere is a wide variety of OPCs ranging from mild to highly toxic.OPCs are structurally different from each other but have a commonmechanism of action, i.e. inhibition of the neurotransmitter enzyme,

Figure 3. Transmission electron microscopy of pancreatic beta cells.Note that the structure of the granules and other cytoplasmic organellesis intact in all the four groups. Magnication: 14 000 .acetylcholine esterase at nerve junctions. Excessive acetylcholineproduces unwanted muscarinic effects (miosis, bradycardia,glandular hypersecretion and gastrointestinal dysfunction) andnicotinic effect, which includes muscular twitching and seizure.

The literature review shows that exposure to OPCs can impairglucose metabolism, through a variety of factors, as highlightedin the introduction and Table 1AD.

This study showed that contrary to previous reports on otherOPCs, POX, at sub-lethal and sub-chronic exposure, with up to49% RBC-AChE inhibition (a mild effect according to Balali-Moodand Balali-Mood, 2008) did not induce diabetes mellitus inexperimental rats. This is the rst report showing that an extremelytoxic OPC, such as POX does not cause diabetes mellitus. Nurulainet al. (unpublished data) worked on another extremely toxic OPCwith the same protocol and observed similar result. Sadeghi-Hashjin et al. (2008) did not attribute any diabetogenic effectto azinphos methyl, a class Ib OPC. However, it must be notedthat the treatment with azinfos-methyl was only for 8 days.The present study was conducted with a sub-lethal dose ofPOX for 6 weeks. Many previous reports have linked exposureto other OPCs such as parathion, a precursor of POX, to thedevelopment of diabetes worldwide (Begum and Rajini, 2011;Kamath and Rajini, 2007; Rezg et al., 2006; Sadeghi-Hashjin et al.,2008; Slotkin, 2011). Other investigators have also shown thatmalathion, dimethoate and other OPCs can cause diabetesmellitus in animal and clinical studies (Table 1AD). It is not clearwhy POX does not cause diabetes in accordance with other OPCs.

J. Appl. Toxicol. 2013; 33: 10361043 Copyright 2012 John

10It is possible that a longer exposure period may predispose theanimals to diabetes, but this has to be thoroughly investigated.Moreover, there is also the possibility that different OPCs causea diabetogenic effect at different periods of exposures. Kuehn(2008) studied the link between an OPC pesticide, dichlorvos,and agriculture workers. He postulated that the incidence of diabe-tes increased with cumulative days of exposure. The unique toxicprole of each OPC may also be a factor. Some OPCs may causediabetes while others do not. Therefore, from the results of thisstudy, it may be concluded that a generalized hypothesis maynot be appropriate to conclude that all OPCs are diabetogenic,whether in acute, chronic or sub-chronic exposure.

The body weight of rats in the POX-treated groups wasslightly but not signicantly lower than the control groups. Adecrease in body weight in the OPC-exposed group is a com-mon phenomenon, which has been reported previously (Begumand Rajini, 2011). The relative decrease in body weight may bebecause of a decrease in appetite or gastrointestinal dysfunctioninduced by OPCs poisoning.

Although the liver/body weight ratio was slightly lower in thePOX-treated group than the control, it was not signicantly differ-ent. The same pattern was observed in monocrotophos-treated(Begum and Rajini, 2011) and diazinon-treated (Ueyama et al.,2007) rats. The lower liver/body weight ratio was attributed toa disturbance in glucose homeostasis and enhanced glucoseoutput (Joshi and Rajini, 2009; Rahimi and Abdollahi, 2007).

The electron microscopy of the islet cells of POX-treated ratsshowed nomorphological changes. The pancreatic beta and otherendocrine cells were intact, suggesting that insulin and otherhormone production was normal in the treated rats. However,Hagar and Fahmy (2002) reported ultrastructural changes in pan-creatic islet cells after 2 months of chronic dimethoate treatment.Hagar and Fahmy (2002) showed that pancreatic beta cells werethe most affected among the damaged islets cells compared withthe control. They showed that most of the beta cells displayed amarked reduction in the number of cytoplasmic granules, withmultiple vacuoles. Gokcimen et al. (2007) observed that thediazinon effect is dose dependent and 1015% of the LD50dose caused fat necrosis, cellular and glandular degeneration.Pournourmohammadi et al. (2007) investigated the effects ofmalathion on insulin secretion from rat pancreatic islets. Their lightmicroscopic examination in semithin sections revealed that mala-thion causes patchy degenerative changes in pancreatic islets ina dose-dependent manner. In terms of toxicity, dimethoate, diazi-non and malathion belong to the mildly toxic group of OPCs andhave been reported to cause diabetes mellitus (Table 1A, B). Theirtoxic effect on pancreatic islet cells is thus obvious. However, thepoint to note is that they produced the effect in a dose-dependentmanner. In this study, the 100 nmol dose was the maximum toler-able dose for the 6-week treatment. Mortality was noted in the rstweek of treatment when 150 nmol was used as the tentative nexthigher dose of POX. However, neither hyperglycemia nor glycos-uria was observed. The absence of morphological alterations inpancreatic beta cells is convincing evidence that POX does notcause diabetes mellitus at sub-lethal chronic exposure.

Urinalysis in the POX-treated group (G2) revealed a normalglucose level compared with the control (G1). However, a smallbut not signicant increase in protein and bilirubin level in urinewas noted in the POX-treated group compared with the control.The presence of protein and bilirubin indicates a dysfunction ofthe kidney and liver, respectively. Liver and kidney disorders canbe caused by OPC (Hayes et al., 1978).Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jat

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2000, 2001), altered level of pre-existing enzymes (Milatovic

insecticide toxicity Short review. Pestic. Biochem. Physiol. 98: 145150.

S. M. Nurulain et al.

1042and Dettbarn, 1996), the modied dynamic properties of theerythrocyte membrane (Testa et al., 1988; Watala, 1993) or analteration in the cholinergic system because of diabetes(Wahaba and Soliman, 1988) may also be possible factors forRBC-AChE inhibition in diabetes.

POX (100 nmol per rat) inhibited the same level of RBC-AChEinhibition (49% of baseline) in diabetic rats compared with thecontrol. This shows that although diabetes is associated with alow level of RBC-AChE, it does not lead to excessive reductionafter the administration of 100 nmol of POX. Moreover, the addi-tion of POX does not exacerbate diabetes mellitus in STZ-treatedrats. This is in contrast to previous reports. According to Begumand Rajini (2011), monochrotofos at a sub-lethal dose, signi-cantly increased the hyperglycemic outcome in STZ-induceddiabetic rats, more than 56% above control rats. Ueyama et al.(2007) reported that diazinon-oxon, a metabolite of diazinon,caused hyperglycemia in STZ-induced diabetic rats. In the pres-ent study, no such effects were observed, suggesting that POXhas a different toxic prole regarding glucose metabolism whencompared with other OPCs.

In conclusion, POX neither induces diabetes mellitus norincreases the glycemic level of diabetes rats indicating thatnot all OPCs are diabetogenic.

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