JOURNAL OF APPLIED TOXICOLOGY, VOL. 11(6), 391-395 (1991)

In Vitro Effects of Organophosphorus Compounds on Calmodulin Activity

I. Pala, P. J. S. Vig and D. Desaiaht Department of Neurology, University of Mississippi Medical Center, 2500 North State Street. Jackson, MS 39216. USA A. Srinivasan Department of Biology, Tougaloo College, Tougaloo, MS 39174. USA

Key word\: organophosphorus compounds; calrnodulin: neurotoxicity: fluorescence

In vitro effects of organophosphorus compounds (OP), such as malathion (M), methyl parathion (MP) and ethyl parathion (EP), on calmodulin (CaM) activity and its active conformation were studied to understand the mechanism(s) of neurotoxicity, since CaM is known to regulate Ca2+ transport and the enzymes involved in signal transduction and nucleotide metabolism. The biological activity of CaM was assessed as a measure of phosphodiesterase (PDE) stimulation. The effect of OP compounds on the active conformation of CaM was determined by studying the binding of fluorescence probes, namely N-phenyl-1-naphthylamine (NPN), and changes in dansyl-calmodulin fluorescence. Dansylated calmodulin was also used to study the effect of OP compounds on complex formation between CaM and PDE. All three OP compounds inhibited the CaM activity and its active conformation in a concentration-dependent manner. Malathion was less effective in comparison to E P and MP, with 1Cs0 values of 37 pM, 34.5 p M and 32 pM, respectively, for CaM activity. EP and MP significantly altered NPN and dansyl-calmodulin fluorescence (50 p M concentrations of OP compounds), whereas M did not show any significant effect on NPN fluorescence. All these compounds significantly affected complex formation between the dansylated CaM and PDE. These results suggest that O P compounds may be interacting with CaM, altering its active conformation, and thus may be inhibiting its biological activity.

INTRODUCTION

Organophosphorus compounds (OP) are the most widely used pesticides by virtue of their biodegradable nature and short persistence. These OP compounds not only produce biological and pathological changes but also cause significant biochemical alterations. All the OP compounds (Phenyl phosphonothioate esters) are uniformly neurotoxic and cause polyneuropathy in humans and chicks;’. the neurotoxicity is due to their ability to inhibit the enzyme cholinesterase,’ particularly in nerve tissue, resulting in an accumulation of acetylcholine.

Calmodulin (CaM) is a calcium-binding. widely distributed, low-molecular-weight (16 700) protein found in high concentrations in mammalian brain.3. It is known to regulate many calcium-dependent processes and a large number of cellular functions.s-x Modification of its activity by toxic compounds and other chemicals could have profound physiological consequences. A variety of antipsychotics and other psychoactive agents’-i and toxicants, including pestic- ides, insecticides and toxic metals,”-i7 have now been shown to interact with Ca”-CaM complexes and alter various CaM-dependent enzyme activities. However, there are marked differences in their affinities for CaM and in their potencies for inhibiting its activity. The Cali -induced conformational change in CaM exposes the hydrophobic groups, which are the binding sites for the target enzymes.ix. I ” CaM antagonists, such

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as phenothiazines and naphthalene sulphonamides, interact with hydrophobic groups of CaM and block its biological activity.’, Like these CaM antagonists, OP compounds may interact directly with CaM and alter its active conformation, thereby altering various cellular processes dependent on CaM and resulting in neuronal dysfunction. In an attempt to understand the molecular mechanism(s), we have studied the effects of three OP compounds on calmodulin activity and its active conformation in vitro.

MATERIALS AND METHODS

Materials

Ethyl parathion (EP), methyl parathion (MP) and malathion (M) were obtained from Pesticide Reposi- tory, US Environmental Protection Agency, Research Triangle Park, NC. All these compounds are of technical grade and > 98% pure. Activator-deficient phosphodiesterase and the fluorescent probes N-phe- nyl-1-naphthylamine (NPN) and dansyl-calmodulin were purchased from Sigma Chemical Co., St. Louis, MO. Bovine brain CaM was purchased from Calbi- ochem Corporation, La Jolle, CA. All other chemicals were analytical grade and commercially available.

Preparation of test solutions

Stock solutions of EP, MP and M were prepared in ethanol. The final concentration of ethanol in the reaction mixture was < 0.5%. This amount of ethanol did not affect either the PDE activity or the direct

0260437X/9 1/06039 1-05$05.00 0 1991 hy John Wiley & Sons. Ltd.

Received I 4 Juiritnry 1991 Accepred I5 April 1991

392 I . PALA ET AL.

binding of NPN to CaM or the displacement of NPN by other compounds.

Phosphodiesterase assay CaM activity (in terms of CaM-dependent PDE stimulation) was determined using the method of Wallace et ~ 1 . ~ ” The reaction mixture contained 40 mM Tris-HCI (pH 8.0), 3 mM MgS04, 50 pM CaC12, 20 ng CaM, 20 pg CaM-deficient PDE and different concentrations of OP (EP, MP and M) compounds. The basal enzyme activity was measured in the absence of CaM. The reaction was initiated by the addition of [‘HICAMP (2 mM containing 200000 cpm per assay) and incubated for 10 rnin at 30°C. The reaction was stopped by placing the tubes in water for 2 min at 95°C followed by an additional 10 min of incubation at 30°C with PDE-free snake venom (1 mg ml-I). The [3H]-adenosine formed was separated by Dowex 1 x 8 anion exchange resin (33% slurry, pH 5.0) and the radioactivity was measured by liquid scintillation coun- ter (Searle, Isocap/300).

NPN fluorescence measurement

The effect of OP compounds on the active conformation of CaM was studied by measuring the changes in NPN fluorescence . I 0 A Perkin-Elmer fluorescence spectrophotometer (Model 650-40) was used to meas- ure NPN fluorescence. The samples were prepared in a total volume of 1 ml containing 50 mM Tris-HCI (pH 7.5) 50 pg CaM, 0.1 mM Ca2+, 1 p M NPN and various concentrations of the test compounds. Excitation and emission intensities were measured at 360 and 420 nm, respectively, with 5 nm slit widths.

Dansyl-calmodulin fluorescence measurements Dansyl-calmodulin fluorescence measurements2’ were made at room temperature in 1 ml of 20 mM Tris-HCI (pH K O ) , 250 mM NaCl, 5 mM MgCl,, 1.18 pM CaM and varying concentrations of test compounds in the presence and absence of 1 pM CaM-deficient phosphodiesterase using the fluorescence spectrophoto- meter. Various concentrations of Ca” were added in order to get the free Ca2+ in the range 0.1-2.0 pM. Concentrations of free Ca2+ were calculated according to Bartfai” using an apparent of 4.4 x loh M-’ at pH 8.0 as reported by Kincaid et All emission spectra were obtained using excitation and emission wavelengths of 340 and 490 nm, respectively, with slit widths of 6 nm.

Statistical analysis Data were expressed as the mean ? SE. All studies requiring statistical evaluation represent a minimum of three experiments in duplicate. Statistical significance was calculated between the control and the activity observed in the presence of test chemicals by using Student’s t-test. A P value of < 0.05 was considered significant. I C ~ ( ) values were calculated by linear regression analysis.

RESULTS

All three OP compounds tested inhibited CaM-depen- dent phosphodiesterase (Fig. 1). The potency was of the order MP > EP > M for CaM-dependent phosphodiesterase activity. The 1cs0 values are tabu- lated in Table 1.

Ca2 ’ -binding to CaM induces a conformational change that exposes a hydrophobic domain; the fluor- escent probe NPN binds to the hydrophobic domain’”. 24. 1 0 and increases the NPN fluorescence intensity. MP and E P decreased the NPN fluorescence in a concentration-dependent manner, whereas M did not show any significant change in NPN fluorescence (Fig. 2). The order of potency was EP > MP. The ~c~~~ values are summarized in Table 1. These icso values are in good correlation with the LDso values reported earlier.

The increase in fluorescence intensity of dansyl- calmodulin as a function of Ca’+ concentration corre- sponds to the occupancy of two high-affinity Ca2 ’

a Ethyl parathion eZa Methyl parathion KQ Malathion

Organophosphates ( 10-50 pM )

Figure 1. Effect of varying concentrations of organophosphorus compounds on calmodulin (CaM) activity. Each value is the mean ? SE of three independent experiments. *Significantly different from control (zero concentration) at P < 0.05.

0-0 E t h y l p a r a t h i o n A-A M e t h y l p a r a t h i o n W-m Mala th ion

Organophosphates (pM) Figure 2. Effect of varying concentrations of organophosphorus compounds on NPN fluorescence. Each value is the mean i- SE of three independent experiments. *Significantly different from control (zero concentration) at P i 0.05.

ORGANOPHOSPHORUS COMPOUNDS AND CALMODULIN ACTIVITY 393

600

600

700

600

500

400

Table 1 . Effects of various organophosphorus compounds on calmodulin activity and on NPN fluorescence”

Compound Calmodulin activity NPN fluorescence Ic50 (PM) IC,, (pM)

-

-

-

.

-

-

Methyl parathion 32.0 Ethyl parathion 34.5 Malathion 37.0

65.01 46.5 -

a The data presented are the mean of three independent experiments.

0

0-0-0 Ethyl parathion 300 I

P) 1.0 1.5 2.0

2 k a,

600 r P) L ; 800 ‘ 700 C

P 0

.r( 4

600 I 0-0

0-P’

M e t h y l parathion _ . v) C 300 1

1.0 1.5 2.0 n

so0 r

800

700

600

500

400 Malathion

300 I 1 .o 1.5 2.0

F r e e Ca2+ ( p M ) Figure 3. Fluorescence intensity of dansyl-calmodulin as a function of Ca2’ concentration. The fluorescence intensity (at 490 nm) of 1.18 p M dansyl-calmodulin was measured in the presence of Ca2+ and in the absence (0-8) and presence (-1 of organophosphorus compounds. The increase in the fluorescence intensity of dansyl-calmodulin occurred over a very narrow free Ca2’ concentration range of 1.5-2.0 pM.26 The concentration of organophosphorus compounds used was 50 kM. Each value is the mean * SE of three independent experiments.

binding sites, whereas occupancy of the third and/or the fourth Ca’+ binding sites results in the subsequent decrease in fluorescence.2h In the presence of phospho- diesterase, the Ca’+-induced fluorescence intensity maxima of dansyl-calmodulin increase further. All three OP compounds at 50 IJ.M concentration decreased the Ca’+-dependent conformational change in dansyl- calmodulin (Fig. 3). The complex formation between dansyl-calmodulin and phosphodiesterase (Fig. 4) also was inhibited by these compounds with similar potency.

DISCUSSION

The present data clearly indicate that the OP com- pounds EP, MP and M at the concentrations tested inhibit CaM-dependent phosphodiesterase activity, suggesting that these OP compounds interact with CaM and thereby impair its biological activity. It is possible that these OP compounds may be interacting with CaM like other organochlorine pesticides,”-IS. ’’ which have been shown to interfere in the activation of CaM-dependent enzymes.

Ca2+ binding to the four Ca’+ binding domains of CaM induces a conformational change that exposes hydrophobic sites. CaM-dependent enzymes and CaM antagonists, such as W-7, chlorpromazine and fluor- escent probe NPN, bind to these hydrophobic sites.’” x, l o CaM antagonists and some organochlorine pestic- ides, such as chlordecone,2x have been shown to suppress the increase in fluorescence of the hydro- phobic probe NPN, induced by complex formation with CaM in the presence of Ca l f . I(’ Since organochlor- ine and OP compounds are lipophilic in nature, these compounds may have a similar type of interaction with CaM. Among the three OP compounds, MP and EP significantly inhibited the Ca’+-dependent increase in NPN fluorescence. This may be due to the differential affinities of MP and EP for the active hydrophobic domain of CaM, or EP and MP may be competing for the NPN binding sites on CaM. All three OP com- pounds inhibited the increase in dansyl-calmodulin fluorescence. The increase in dansyl-calmodulin fluor- escence results from binding of Ca” to two high- affinity CaZ+ binding sites of CaM.25 The Ca2+- dependent (involving the high-affinity sites) complex formation between dansyl-calmodulin and phosphodie- sterase, which further increase the fluorescence inten- sity,” was also inhibited by OP compounds.

These data suggest that OP compounds interact with CaM and induce a conformational change, thereby bringing about an altered response on its target enzymes. The differential effects of MP, EP and M on CaM may result in the altered regulation of many vital neuronal processes, and may partially constitute their neurotoxicity. Since organophosphates are also known to inhibit non-CaM-regulated systems such as the activity of acetylcholinesterase,’” the decrease in CaM activity may be additive to the organophosphate-related neurotoxicity .

Acknowledgements This work was supported by NIH-A126357 and GM08110. 1. P. was a Postdoctoral Fellow supported in part by the Government of India.

394 I. PALA ET AL.

V-V Control 0-0 Ethyl parathion A-A Methyl parathion .-W Malathion

300 I U

1.0 1.5 2.0

Free Ca2+ ( pM ) Figure 4. Effect of organophosphorus compounds on complex formation between dansyl-calmodulin and phosphodiesterase. The fluorescence intensity of 1.18 pM dansyl-calmodulin was measured in the presence of varying concentrations of Ca2+ and 1.0 p M phosphodiesterase. The concentration of organophosphorus compounds used was 50 pM. Each value is the mean t SE of three independent experiments.

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