Influence of pesticide exposure on
carbonic anhydrase II from
sheep stomach
Namık Kılınc¸
1, Mehmet Mustafa _Is¸go¨r
1,
Bu¨lent S¸engu
¨ l
1and S¸u
¨ kru¨ Beydemir
1Abstract
Carbonic anhydrase (CA) is a widely distributed enzyme and has a crucial role in the cells, tissues and organs of living organisms. It is found that CA-II is one of the most abundant CA isoenzymes in the gastrointestinal system. It plays an important role in the gastric acid secretion in stomach. In this study, we purified CA-II iso-enzyme from sheep stomach with a 615.2 purification fold, 78% purification yield and 5562.02 specific activity. Moreover, the in vitro effects of some commonly used pesticides including chlorpyrifos, cypermethrin, dichlor-vos, glyphosate isopropylamine and lambda cyhalomethrin on the enzyme activity were investigated. Of these compounds, glyphosate isopropylamine and dichlorvos showed an inhibition on CA-II esterase activity. They have IC50values of 0.155 mM and 2.690 mM and Kivalues of 0.329 mM and 3.654 mM, respectively. Both
gly-phosate isopropylamine and dichlorvos inhibited CA-II isoenzyme in a noncompetitive manner. Keywords
Carbonic anhydrase, pesticides, sheep stomach, enzyme inhibition
Introduction
Carbonic anhydrase (CA, EC 4.2.1.1) is a zinc metal-loenzyme that catalyzes reversible reaction of carbon dioxide and water to bicarbonate and protons: CO2þ
H2O $ HCO3 þ Hþ. CA (carbonate hydrolyase,
E.C.4.2.1.1) has been found in all living organisms and includes Zn2þin its active site. It is first detected in bovine erythrocytes (Supuran and Scozzafava, 2001). CAs have an important role in a number of different metabolic processes such as acid–base regu-lation, respiration and transportation of carbon diox-ide and bicarbonate, homeostasis, bone resorption, calcification, electrolyte secretion, and so on (Cheg-widden et al., 2000; Supuran et al., 2000).
Until now, 16 different CA isozymes have been detected in animals and plants. CA-I, -II, -III, -VII and -XIII are cytoplasmic, CA-V is mitochondrial, CA-VI is secretory, CA-IV, -IX, -XII and -XIV are mem-brane associated, and CA-VIII, -X and -XI are nonca-talytic isoenzymes (Chegwidden et al., 2000; Dodgson, 1991; Fujikawa et al., 1999; Pastorekova et al., 2004; Supuran et al., 2002; Tashian et al., 1991). HCA-II is the most studied form of CA
isozyme. It has a wide distribution in the organism and is located in many different organs and cell types. Osteoporosis, renal tubular acidosis and cerebral calcification are associated with this isozyme, thus it has a great importance in bone, brain and kidney (Lindskog, 1997). CA isozymes are also found in sig-nificant quantities in the stomach, because they are related with gastric acid secretion in this organ. Parie-tal cells are responsible for the gastric acid production in the stomach. Hydrochloric acid production mechanism in these cells depends on the hydration of carbon dioxide to carbonic acid. This reaction is catalysed by CA enzyme in parietal cells (Davies and Edelman, 1985). These cells contain five times as
1
Biochemistry Division, Faculty of Sciences, Department of Chemistry, Atatu¨rk University, Erzurum, Turkey
Corresponding author:
S¸u¨kru¨ Beydemir, Biochemistry Division, Faculty of Sciences, Department of Chemistry, Atatu¨rk University, Erzurum 25240, Turkey.
Email: [email protected]
Toxicology and Industrial Health 2015, Vol. 31(9) 823–830 ©The Author(s) 2013 Reprints and permissions:
sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233713475508 tih.sagepub.com
much of CA enzyme as that of red blood cells (Daven-port, 1939) (Figure 1).
Pesticides are substances that are used to kill or control the organisms considered to be harmful. The most widely used ones are insecticides, herbicides, fun-gicides and rodenticides. They are the most common contaminants of the environment and cause water, soil and air pollution. They can also accumulate in the living organisms like humans, animals, and so on (Anwar, 1997). It is known that many compounds, metal ions and pesticides can be dangerous for the liv-ing organisms. Scientific studies on pesticides clarify that these compounds show their toxic effects by inhi-biting the enzymes even at low concentrations (Ceyhun et al., 2010; Ekinci et al., 2007a; Gulcin et al., 2004). Chlorpyrifos and dichlorvos are organophosphate insecticides that are highly toxic substances and inhibit acetylcholinesterase in the metabolism. Cypermethrin and lambda cyhalomethrin are pyrethroids and used as strong neurotoxins for insects. Glyphosate isopropy-lamine is a herbicide and used widely to kill weeds. It shows its effect by inhibiting an enzyme used in the synthesis of aromatic amino acids.
In the present study, CA-II isoenzyme was purified from sheep stomach in single step using sepharose-4B– L-tyrosine–sulfanilamide affinity chromatography, and we investigated the in vitro effects of some commonly
used pesticides including chlorpyrifos, cypermethrin, dichlorvos, glyphosate isopropylamine and lambda cyhalomethrin on the purified isoenzyme.
Material and methods
Materials
Sepharose-4B, protein assay reagents and chemicals for electrophoresis were purchased from Sigma-Aldrich (Taufk irchen, Germany). All other materials used in the study were obtained from Merck (Darmstadt, Germany) and of analytical grade.
Homogenate preparation
Sheep stomach used in the experiments was obtained from fresh the slaughter. Sheep stomach was pre-served in ice and brought to the laboratory. They were cut into pieces of 20 g and stored at80C for later use. Tissue samples of blood and other contaminants were eliminated with 0.9% NaCl by washing 3 times. To prepare tissue homogenate, the sample was cut into very small pieces using a knife, and then thawed using liquid nitrogen and homogenized in buffer solution of 3 ml/g, 25 mM Tris HCl/0.1 M Na2SO4
(pH ¼ 8.7). This suspension was centrifuged at 15,000g for 30 min. Supernatant was taken and
CO2 + H2O + H+ Carbonic Anhydrase Parietal cells Cl– Cl– H+ HCO3– HCO3– HCO3– H+ K+ K+ Cl– Cl– Blood Gastric lumen Figure 1. Role of carbonic anhydrase in parietal cells of stomach.
ultracentrifugation was performed at 100,000g. The supernatant was used for next analysis.
Purification of CA from sheep stomach by affinity
chromatography
The pH of the homogenate obtained from sheep sto-mach was adjusted to 8.7. Homogenate was applied to the column and washed with a solution of Tris-HCl/22 mM and 400 ml of 25 mM Na2SO4 (pH
8.7). Thus, the enzyme CA was attached to the col-umn and other impurities were removed. Then, 25 mM Na2HPO4/1 M NaCl (pH 6.3) buffer was
applied to the column to eluate CA-I enzyme. After that CA-II enzyme was eluted by applying 0.1 M NaCH3COO.3H2O/0.5 M NaClO4 (pH 5.6) solution
to the column. Flow rate through the column was adjusted to 20 ml/h, with the help of a peristaltic pump. Eluates were taken into the tubes with a frac-tion collector and absorbance of the tubes was mea-sured at 280 nm for qualitative protein determination.
Dialysis
Tubes showing high absorbance at 280 nm were grouped separately, placed in dialysis bags and dia-lysed for 2 h against a buffer (containing 1 mM b-mercaptoethanol and 0.05 M Tris-SO4, pH 7.4).
Dia-lysis procedure was performed in cold environment.
Measurement of CA enzyme activity
CA enzyme activity can be measured in two ways: first one is CO2-hydratase activity, the physiological
activity of the CA, and the second one is esterase activity that can be carried out in vitro and followed spectrophotometrically. Enzyme hydratase activity measurements performed according to the method described by Wilbur and Anderson (1948). This activity was calculated in terms of enzyme unit (EU) using the equation: (t0 tc)/tc, where t0 and
tcare the times for pH change of the nonenzymatic
and the enzymatic reactions, respectively. CA enzyme uses p-nitrophenylacetate as the substrate of hydrolysis of p-nitrophenol and acetate in in vitro cases. Esterase activity of the enzyme was assayed as increase in absorbance at 348 nm.
Protein determination
Quantitative amount of protein for the purified enzyme solution was measured spectrophotometri-cally at 595 nm according to Bradford’s method and
bovine serum albumin is used as a standard (Bradford, 1976).
Sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE)
After the purification of CA enzymes from sheep sto-mach, purity of the enzyme was verified by 3%–8% discontinuous sodium dodecyl sulfate polyacrylamide gel electrophoresis (Laemmli, 1970) (Figure 2).
In vitro studies
Inhibitory effects of some commonly used pesticides on CA enzyme activity purified from sheep stomach were tested in triplicate at each concentration used. CA activities were measured in the presence of differ-ent concdiffer-entrations of inhibitors. Control activity was assumed to be 100% in the absence of inhibitor. For each compound, a percentage activity versus inhibitor concentration graph was drawn. For determination of Ki values, three different inhibitor concentrations
Figure 2. Sodium dodecyl sulfate–polyacrylamide gel elec-trophoresis (SDS-PAGE) analysis of purified CA II. Lanes 1 and 2: CA II from sheep stomach via Sepharose 4B–L
-tyro-sine–sulfanilamide affinity chromatography. Lane 3: stan-dard proteins (kDa): Escherichia coli b-galactosidase (116), rabbit phosphorylase B (97.4), bovine serum albu¨min (66), chicken ovalbumin (45) and bovine carbonic anhy-drase (29) were used as standards.
were tested at five different substrate concentrations for each chemical. Lineweaver–Burk curves were used for the determination of Ki value and type of
inhibition (Lineweaver and Burk, 1934).
Results
CA-II isozyme from sheep stomach was purified using a simple method in only one step using Sephar-ose-4B–L-tyrosine–sulfanilamide affinity column. Enzyme was purified by a 615.2 purification fold, 78% purification yield and 5562.02 specific activity (Table 1).
Figure 2 shows the sodium dodecyl sulfate–polya-crylamide gel electrophoresis (SDS-PAGE) photo-graph performed to verify the purity of the enzyme and calculate the molecular weight of it. The molecular weight of enzyme was determined as approximately 29 kDa, in this method.
Inhibitory effects of pesticides were tested under in vitro conditions. For the compounds showing inhibi-tory effects, the IC50 value was determined by
activity% vs pesticide concentration graphs for each compound. Two pesticides, glyphosate isopropylamine and dichlorvos, inhibited CA-II esterase activity signif-icantly. The IC50values of the compounds were found
to be 0.155 mM and 2.690 mM, respectively. Linewea-ver–Burk curves were used to determine the Ki value
and the type of inhibition of the compounds. Three ferent inhibitor concentrations were tested at five dif-ferent substrate concentrations for each compound. Ki
values were calculated as 0.319 mM and 3.700 mM on CA-II esterase activity. Both glyphosate isopropyla-mine and dichlorvos inhibited CA-II isoenzyme in a noncompetitive manner (Table 2 and Figure 4).
Discussion
CA is a member of an important class of zinc metal-loenzyme family and plays a vital role in regulating CO2 levels in living organisms (Beydemir et al.,
2000; Esposito et al., 2000). In metabolism, CA iso-zymes have many physiological functions like inter-conversion of CO2 and HCO3, physiological pH
control and gas balance, calcification, photosynthesis and also ion transport and pH regulation in eye, kid-ney, central nervous system (CNS) and inner ear (Beydemir et al., 2002). In stomach, CA exists abun-dantly in parietal cells (oxyntic cells) and catalyzes the reaction between carbon dioxide and water to form carbonic acid and takes part in the metabolism of gastric hydrochloric acid production (Davenport, 1939; Davies and Edelman, 1985). In our study, CA-II isoenzyme was purified from sheep stomach using sepharose 4B–L-tyrosine–sulfanilamide affinity chromatography. CA-II was purified with a 615.2 pur-ification fold, 78% purpur-ification yield and 5562.02 specific activity (Table 1).
Recently, CA-II is purified from many different sources including human erythrocytes (Beydemir et al., 2002), fish gills (Bone et al., 1995) and fish erythrocytes, rainbow trout’s (RT) brain and liver (Soyut and Beydemir, 2008; Soyut et al., 2008), RT’s kidney (Soyut and Beydemir, 2011) and rat sal-iva and rat erythrocytes (Feldstein and Silverman, 1984). Our SDS-PAGE result is compatible with those of the mentioned studies. It can be seen from Figure 2 that the enzyme has a single band that veri-fies the enzyme purity, and its molecular weight is approximately 29 kDa in the results of SDS-PAGE technique (Figure 2).
Table 1. Summary of purification procedure of carbonic anhydrase (CA)-II from sheep stomach. Purification step Activity (EU/ml) Total volume (ml) Protein (mg/ml) Total protein (mg) Total activity (EU) Specific activity (EU/mg) Purification factor Yield (%) Homogenate 65 40 7,07 282,8 2600 9,19 1 100 Sepharose-4B–L tyrosine– sulfanilamide affinity chromatography and dialysis 290 7 0,0513 0,3591 2030 5652,02 615,2 78
Table 2. IC50, Kivalues and inhibition types of pesticides on the enzyme activity.
Pesticide IC50 mM Average KimM Inhibition type Glyphosate isopropylamine 0.155 0.319 + 0.0673 Noncompetitive Dichlorvos 2.69 3.700 + 1.67 Noncompetitive
It has been known that almost all chemical reac-tions in the metabolism of the living organisms are catalyzed by enzymes, and the enzyme activities are influenced (decreased or increased) by many chemi-cal substances including pesticides, fungicides, drugs and metal ions even at low concentrations (Alici et al., 2008; Ekinci et al., 2007a, 2007b). There are large numbers of studies about inhibition or activation of CA enzyme activity with various chemical substances and drugs reported in the literature (Beydemir and Gu¨lc¸in, 2004; Beydemir et al., 2000; Supuran et al.,
2001). For example, Ekinci and Beydemir investi-gated the inhibitory effects of some pesticides and fungicides on the enzymatic activity of RT’s CAs, and they found that deltamethrin inhibits the enzyme at very low doses, both in vitro and in vivo (Ekinci and Beydemir, 2010). In another study, Ceyhun et al. stud-ied the inhibitory effects of some pesticides on the enzymatic activity of RT’s CA. Their results indi-cated that deltamethrin, diazinon, propoxur and cypermethrin inhibited CA activity in vitro, and the most effective one is deltamethrin which is widely
P N Cl l C l C O S O O Chlorpyrifos P O O O O Cl Cl Dichlorvos Cl Cl O O N O Cypermethrin OH O NH P HO OH O Glyphosate isopropylamin O O N O Cl F F F Cyhalothrin
Figure 3. Structures of the pesticides used in this study: chlorpyrifos, dichlorvos, cypermethrin, glyphosate isopropyla-mine and cyhalothrin, respectively.
used both at homes and in agricultural fields. Delta-methrin inhibited the enzyme at very low doses, par-ticularly in vivo (Ceyhun et al., 2010).
From the above mentioned studies, it can be clearly seen that so many pesticides have inhibitory effects on CA enzyme activity. Therefore, in our study we inves-tigated the inhibitory effects of some widely used pes-ticides on the activity of purified CA-II isoenzyme (Figure 3). From the pesticides used in the study, gly-phosate isopropylamine and dichlorvos inhibited CA-II esterase activity; they have no meaningful effect on CA-II hydratase activity. Other pesticides, chlorpyrifos, cypermethrin and lambda cyhalome-thrin were found to have no significant effect on CA-II enzyme esterase and hydratase activities. The IC50values of glyphosate isopropylamine and
dichlor-vos for CA-II esterase activity were found to be 0.155 mM and 2.690 mM, respectively (Table 2 and Figure 4). Glyphosate isopropylamine and dichlorvos were found to have a Ki value of 0.329 mM and
3.654 mM on CA-II esterase activity. Both glyphosate
isopropylamine and dichlorvos inhibited CA-II isoenzyme in a noncompetitive manner (Table 2 and Figure 4).
Glyphosate is the most widely used pesticides in agriculture and the second most in homes and gar-dens. It is marketed in more than 100 countries world-wide, and the annual running cost of this pesticide in the United States is approximately 103–113 million pounds (Kiely et al., 2004). Based on our in vitro stud-ies regarding the effects of pesticides on the activity of CA II, Glyphosate was found to be the most effec-tive on the enzyme activity inhibition. Moreover, this substance indicated 10 times lower IC50 value than
dichorvos. This result means glyphosate, a very widely used pesticide all over the world, inhibited enzyme activity by half even at 10 times lower con-centrations than dichlorvos.
Inhibitors mostly show their effects by changing the activity of the enzyme in the metabolism. IC50
value is the concentration of the inhibitor that decreases the enzyme activity by 50%. Smaller IC50 y = 100e–0.2613x R2 = 0.9552 y = 100e–4.5284x R2 = 0.9313 0 20 40 60 80 100 120 0 1 2 3 4 5 6 –1 1 3 5 7 9 Dichlorvos (µM) Percentage activity 0 20 40 60 80 100 120 Percentage activity 0 0.05 0.1 0.15 0.2 0.25 0.3 0.5 2.5 4.5 6.5 8.5 Glyphosate isopropylamin (µM) 0 500 1000 1500 2000 2500 1/V
Control [I]=0,071 µM [I]=0,142 µM [I]=0,284 µM
0 500 1000 1500 2000 2500 3000 3500 4000 –1.5 1/S 1/S 1/V
Control [I]=1,24 µM [I]=3,1 µM [I]=3,72 µM
(a)
(c) (d)
(b)
Figure 4. Determination IC50and Kivalues of the pesticides, dichlorvos and glyphosate isopropylamine: (a) IC50graph of dichlorvos, (b) IC50graph of glyphosate isopropylamine, (c) Kigraph of dichlorvos and (d) Kigraph of glyphosate isopro-pylamine. For the determination of Ki values, three different inhibitor concentrations were tested for each drug. P-nitrophenylacetate was used as substrate at five different concentrations. Control activity was assumed to be 100% in the absence of inhibitor. Activity assays were performed as described in Materials and Methods.
values indicate that enzyme activity can be inhibited to its half at lower concentrations. Indicators having smaller IC50 values are stronger inhibitors. In our
study, glyphosate isopropylamine and dichlorvos inhibited CA-II isoenzyme at micromolar level. These values show they are very effective inhbitors of this enzyme. Even small dosage of these compounds can affect the metabolism of living organisms. Similar studies have shown that pesticides can inhibit CA enzyme because of electronegative atoms in their structures (Ceyhun et al., 2010; Dogan, 2006). Of the pesticides that were used in our study, only glypho-sate isopropylamine and dichlorvos showed signifi-cant inhibitory effects on the enzyme. Both of these compounds have phosphate groups in their structures. It is estimated that phosphate groups of these com-pounds played an important role in the inhibition mechanism of the enzyme. Both of the pesticides inhibited the enzyme noncompetitively. In noncom-petitive inhibition, inhibitor shows its effect by bind-ing enzyme at a site other than the active site. Presumably, the phosphate group of glyphosate iso-propylamine and dichlorvos interacts with enzyme amino acid residues away from the active site and causes noncompetitive inhibition.
In conclusion, we purified CA enzyme from sheep stomach using a simple method and investigated the inhibitory effects of some commonly used pesticides. Glyphosate isopropylamine and dichlorvos were found to have the highest inhibitory effects on the pur-ified enzyme. Because of their toxicity, pesticides affect not only the target organisms but also animals and humans which are being exposed to these com-pounds. So, some measures have to be taken to pre-vent excessive diffusion to the environment when using these compounds. Also, potential health effects of these pesticides must be well considered and the usage of them must be kept under control.
Funding
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
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