Carbofuran abrogates the membrane Ca + +-ATPase activity in liver : the role of cholesterol

Carbofuran is a broad spectrum pesticide used in agricultural fields and domestic places throughout the world. It is one of the deadly toxic carbamate pesticide that kills the pest by inhibiting the crucial enzyme of nervous system known as acetyl cholinesterase. In the present study, we report how carbofuran increases the different spectrum of cholesterols, including free cholesterol and esterified cholesterol in the fish hepatocytes. It is observed that induced-cholesterol can inhibit the enzymatic activity such as Ca++ATPase, which is a critical protein for maintaining the calcium homoeostasis in the cellular microenvironment. Carbofuran integrates into human body through foods and drinks. As trace of carbofuran is identified in our daily food and drinks, we examined the homology of Ca++-ATPase between the fish and human, so our data can illuminate the effects of carbofuran on this crucial enzyme. While studying the homology with the help of bioinformatics, we recognized that there is around 70% homology in the protein sequence of Ca++-ATPase between fish Heteropneustes fossilis and human (Homo sapiens), which appears as sufficient to simulate our fish-model data in human. This study demonstrates that carbofuran affects our day-to-day life by inhibiting Ca++-ATPase through modulation of lipid synthesis, a critical regulatory system that controls overall homeostasis in our body.


Introduction
Carbofuran (trade name is furadon) is one the most commonly used pesticide in agricultural fields and domestic places [1]. The mechanism of its toxic action is mediated through irreversible binding of a crucial enzyme of nervous system known as acetyl cholinesterase, which breaks the acetylcholine in acetic acid and choline in physiological system [2]. The extended half life of carbofuran on the soil ecosystem makes it more venerable to disperse from one ecosystem to other [1]. Surprisingly, traces of carbofuran were detected in the foods and drinks, which is consumed day-to-day in our life [3,4,5]. The biochemical structure of carbofuran is similar to steroid hormone with an organic benzene nucleus which makes it a more lipophilic compound [6]. New study revealed that carbofuran can disrupt the steroid hormone homoeostasis by binding its receptor and inducing a mock signaling [1]. Abnormal sperm count was observed upon intake of carbofuran in rat model [7].
Chronic exposure to this pesticide seemed to exert a carcinogenic effect on the baby in mother's womb [5,7,8]. A mood disorder was observed to the patients, which might be due to its ability of inhibiting to acetyl cholinesterase, a critical enzyme in our nervous system [9]. The acute toxicity of carbofuran is fatal to human and animals if exposed in high dose. Thousands of birds and wild animals die due to ingestion of granular form of carbofuran in agricultural fields [10].
The detoxification of carbofuran occurs in the liver. Our laboratory reported that cytochrome P4501a might be involved in degradation of this pesticide in hepatocytes [11,12,13]. The mechanism of degradation is also associated with the induction of several critical pathways including, PI3 kinase, PKC, and HSP70 [12].
There are lots of report indicated that, upon metabolism, carbofuran produces 3-keto carbofuran and 3-hydroxycarbofuran [14]. The metabolic biproducts of carbofuran are relatively water soluble and therefore excrete from our body, however; there is no knowledge about the cellular compartmentalization of these products in the hepatocytes while it undergoes detoxification. In this manuscript, we have identified its different metabolites in subcellular fractions upon detoxification, such as, nucleus, cytosol, mitochondria, and membrane by high pressure liquid chromatography (HPLC). Membrane Ca++-ATPase is one of the critical enzymes that work in the endoplasmic reticulum (ER) and maintain the homeostasis of calcium inside the cell [14,15]. Disruption of Ca++-ATPase leads to dysregulation of many signaling pathways which may result in apoptosis of cells [16]. Here we observed that carbofuran inhibits the Ca++-ATPase activity dose dependently in the cultured catfish hepatocytes. While investigating the homology of Ca++-ATPase between fish Heteropneustes fossilis and human Homo sapiens, we observed that there is around 70% homology between these two species, indicating that our data can be simulated with human system. Few earlier reports indicated that pesticides have the ability to disrupt the Ca++-ATPase activity, however, there was no clear mechanism [17,18,19]. While searching the underlying molecular mechanism, we observed that carbofuran induced the spectrums of cholesterol in the hepatocytes membrane. The elevated cholesterol could be responsible for the attenuation of membrane Ca++-ATPase activity. Our observations revealed a new mechanism of carbofuran toxicity, which is one of most useful pesticides being utilized nowadays to save crops and domestic products. The data also signify that carbofuran when enters our body through food and water could inhibit the Ca++-ATPase via modulation of lipid synthesis or its metabolism, and affects the overall Ca++ homeostasis in liver.

Materials:
Carbofuran ( catfish liver and their culture were done following the methods routinely used in our laboratory [11].

Treatment of the hepatocytes with carbofuran (CF)
Carbofuran was added at a concentration of 1 uM to the culture media and incubated for 24h. Treated hepatocytes were harvested and pooled for further experiments. Pellet was obtained from harvested hepatocytes upon centrifugation at 500 x g for 5 minutes followed by measuring the wet-weight of hepatocytes.
Pellet was sonicated by a sonicator (Proscientific, oxford, CT ) in 20 mM of Tris buffer with 6 strokes for 10 sec each. Nucleus, mitochondria, cytosol, and membrane were separated by differential centrifugation. Nucleus was obtained at 500 x g, where as mitochondria was obtained at 10,000 x g from post nuclear supernatant.
Membrane was obtained by ultracentrifugation at 100,000 x g for 1 h. The supernatant was collected as a cytosol. Those four fractions were freezed down at -80ºC for carbofuran and its metabolites extraction.

Preparation of samples for HPLC
Extraction of carbofuran and its metabolites was performed following the method of Chatterjee et al. [20,21]. The fractions containing carbofuran and its metabolites were cleaned by passing through a column The staining reagents were sprayed uniformly on the plate followed by heating at 100-110ºC for 20 minutes.
Cholesterol were detected as a violet spot on the TLC plate. The stained spots were quantified by a densitometer against the standard using the software (Bio-Rad 700-GS, Imaging Densitomter, Japan).

Analytical estimation of cholesterol from total lipid
The spectrum of cholesterols from the total lipid was estimated. Total cholesterol, free cholesterol, and esterified cholesterol were determined separately following the method mentioned earlier [23,24].
Assay of membrane Ca++-ATPase from the hepatocytes Ca++-ATPase activity was measured from the hepatocytes membrane following the method described earlier [25,26].

ATPase activity
Insertion of cholesterol in the hepatocytes membrane was performed according to the method of Warren et al. [27]. irreversibly [27]. Ca++-ATPase activity from cholesterol inserted membrane was measured as mentioned before.

Identifying the homology of Ca++-ATPase enzyme
The homology of protein sequence was examined using the pairwise sequence alignment tool from European Bioinformatics Institute (Clustal W and Clustal X, Version -2 ) following the method as mentioned before [28].

Statistical Analysis of the data
Each experiment was repeated at least three times using multiple samples. Statistical analysis of the data was done by Student's t-test between two groups and Duncan's Multiple Range Test among various groups.
Significance level was considered at p<0.05.
(Continued on page 6) its metabolites were found to be more in membrane compared to cytosol, mitochondria, and nucleus (Fig. 2).
Carbofuran remained almost double in membrane compared to cytosol and mitochondria ( ATPase activity occurred in the membrane when exposed to higher amount of carbofuran indicating an involvement of cellular mechanism regulating this crucial enzymatic activity. The activity showed an optimum level at untreated cell followed by gradual inhibition and reduced to minimum at 10 uM of carbofuran (Fig. 3).
This result induced our interest to search the possible mechanism underlying the inhibition of this enzymatic activity.  (Fig. 4C).

ATPase activity through activation of cholesterol.
Findings of previous workers along with our current data indicated that cholesterol might be involved in modulation of membrane Ca++-ATPase activity. In order to examine that, we biochemically inserted cholesterol into the cell membrane followed by  Nucleus, mitochondria, cytosol, and membrane were isolated from sonicated cells by differential centrifugation. CF and its metabolites were extracted from these cellular fractions as mentioned in 'Materials and methods'. Retention time and area of the graph in terms of their amount from HPLC column were shown underneath the picture for each sample as a table. Amount of CF and its metabolites were detected from its standard curve presented in Figure 1. The combined results were presented in Table-1  NS, indicated the value was not significant compared to control.

Freely Available Online
Running title: Carbofuran abrogates Ca++-ATPase

Figure 4: Estimation of cholesterol from carbofuran treated hepatocytes by thin layer chromatography (TLC) and effect of cholesterol on membrane Ca++-ATPase. A.
Cholesterol was extracted from the membrane of carbofuran treated hepatocytes. Thin layer chromatography was performed to quantify the extracted cholesterol and chromatogram was developed. B. Spots were collected from the TLC plate and amount of cholesterol was estimated. * indicated the value was significant compared to control (p<0.001). C. Total, free, and esterified cholesterol were estimated from treated hepatocytes. * indicated the value was significant compared to corresponding control (p<0.05). D. Cholesterol was inserted in the catfish hepatocytes with the help of Na-cholate followed by measurement of Ca++-ATPase activity. * indicated the value was significant compared to control (p<0.05).** indicated the value was significant compared to control (p<0.01). NS, indicated the value was not significant. sapiens. The data demonstrated that there was a homology of 69.7% between these two species (Fig. 5).  [11,13].

Discussion
The deposition of carbofuran in the membrane lipid bilayer caused the elevation of membrane cholesterol level, which was assured by decrease in the Ca++-ATPase activity. CF-induced elevation of cholesterol in the liver of fish was reported by many workers [20,32].
The fish treated with carbamate pesticide showed the higher lipase activity, which might result the mobilization of fatty acids and glucerol from tissue fat depot.
Cholesterol is the necessary compounds for the synthesis of many other compounds. Gupta  During course of evolution the protein sequence of Ca++-ATPase stays almost conserved; however, it was diversified in different organs of human body as isoforms [42]. The 70% homology in the protein sequence between fish and human indicated that the derogatory effect of carbofuran, which was shown in our current investigation, could be extended in human liver as carbofuran was reported to be ingested through foods and drinks in our day to day life. However, this interpolation of the Ca++-ATPase data was drawn based on global protein sequences of this enzyme of fish and human; it would have been strengthened if we could perform the comparative homology of either specific cell type versus cell type, or organ versus organ between these two organisms. This is one of the limitation of our study.

Conclusions
Our data showed that carbofuran treatment increased total, free, and esterified cholesterol, which in turn attenuated Ca++-ATPase activity in the liver.