Studies on Paraoxonase-1 Isolated from Amniotic Fluid and its Effect Against Cisplatin-Induced Hepatotoxicity and Cardiotoxicity in Rats

Paraoxonase-1 (PON1) was isolated from the amniotic fluid of normal pregnancies at last week of gestation. One proteinous band had been isolated by gel filtration sephadex )G-50) from the protein precipitate produced by ammonium sulfate saturation (67%) after dialysis. The product from (G-50) gave two bands by sephadex (G-100). It was found that the first peak (Peak A) had higher activity for (PON1). The apparent molecular weights of the isolated PON1 using gel filtration chromatography and SDS-PAGE was (43873 + 350) and (43682 + 278) Dalton respectively. The results also showed that the optimum conditions of PON1 was obtained at (80 μg/ml) of protein as a source of the enzyme using (10 mmol/l) of paraoxon as a substrate, Tris-HCl buffer (0.14 mol/l) as a buffer at pH (8.0) and incubation for (7) minutes at (45C). Using lineweaver– Burk plot, the values of maximum velocity (Vmax) and Michaelis constant (Km) were (80.0 μmol/ min) and (3.79 mmol/l) respectively. The protective effect of PON1 against cisplatin-induced hepatotoxicity and cardiotoxicity by intraperitoneal injection of (10mg/kg) cisplatin were evaluated in 35 male albino white rats classified into 5 groups. The rats were treated with 0.5mg/kg/day or 1mg/kg/day of isolated PON1 injected intraperitonealy for 5 successive days before and 5 successive days after induction of toxicity. The results showed a significant reduction in the levels of alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), total bilirubin(TB) and malondialdehyde(MDA) in comparison with the cisplatin treated animals. It was concluded that (PON1) protects the liver and heart against the toxicity induced by this cytotoxic drug.


INTRODUCTION
Paraoxonase-1 (PON1, EC 3.1.8.1) is a Calcium dependent mammalian enzyme that is synthesized primarily in the liver and is secreted into the serum where it is associated with high density lipoproteins (HDLs) and has a protective effect against the oxidation of low density lipoproteins (LDLs). Beside antioxidant and antiatherogenic properties, PON1 also is a detoxifier that can hydrolyze toxic organophosphates (Sanghera et al., 1998). It is a member of three gene family consisting of PON1, PON2 and PON3 located on human chromosome 7 (Primo-Parmo et al., 1996). Several studies have shown that PON1 can bind reversibly to organophosphate substrates which it hydrolyze (Arslan et al., 2011 ). Environmental factors that change PON1 activity include tobacco consumption, which has been reported to depress PON1 activity and concentration (Mackness et al., 1996). Human paraoxonase (PON) is believed to protect lipoproteins against oxidative modification (Gowri et al., 1999).
Amniotic fluid (AF), the protecting liquid contained in the amnion cavity, is an essential component for fetal development and maturation during pregnancy. It also plays a significant defensive role as part of the innate immune system since AF has an organized pool of antimicrobial peptides against common bacterial and fungal pathogens (Akinbi et al., 2004). AF is composed of 98% water and electrolytes, proteins, peptides, carbohydrates, lipids and hormones, Human AF has been shown to contain 412 proteins (Tsangaris et al., 2006), It provide the physical environment, which protects the growing fetus (Verburg et al., 2007). Human phosphate binding protein is an apolipoprotein, 38-45-kDa glycosylated protein, found in human AF (Tsangaris et al., 2006) for example PON1 (Alam et al., 2009).
Cisplatin is a platinum-based drug (Ali and Al Moundhri, 2006), which is one of the most effective anti-neoplastic agents used for treatment of testicular, ovarian, bladder, cervical, lung, and neck cancers (Abu-Surrah and Kettunen, 2006). The cytotoxic effect of cisplatin is believed to result mainly from its interaction with DNA, via the formation of covalent adducts between certain DNA bases and the platinum compound (Yousef et al., 2009). However, despite it's a clinical usefulness, cisplatin treatment has been associated with several toxic side effects including nephrotoxicity (De Jongh et al., 2003), hepatotoxicity and cardiotoxicity (Al-Majed, 2006).
The aim of this research is to provide a detailed study of (PON1) involving isolation, purification from AF using different biochemical techniques then characterization of PON1 and showing its effects on hepatotoxicity and cardiotoxicity in rats induced by cisplatin drug.

MATERIALS AND METHODS Assay the activity of plasma paraoxonase-1 (PON1)
PON1 assays were done by formation of p-nitrophenol and measured by its absorbance at 405 nm for 5 min (Equation Below). Assay buffer contained 0.125 mol/l Tris-HCl (pH 8.5), 1.25 mmol/l CaCl 2 , and 1 mol/l NaCl (pH 8.5) (Eckerson et al., 1983;Furlong et al., 2001). For each set of assays, 6 mmol/l freshly prepared paraoxon(O,O diethyl-O-p-nitrophenylphosphate; Sigma-Aldrich Chemical Co.) substrate solution of 120 mmol/l paraoxon in acetone diluted with 0.125 mmol/l Tris-HCl was used. Paraoxon stock solution was handled very cautiously with protective measures. The assay tube contained 750 μL of Tris buffer, 50 μL of sample (1:2 diluted with water) and 200 μL of 6 mmol/l paraoxon. The reaction was initiated at 37°C by the addition of the substrate solution, and using a CE 1021 Ultra Violet and Visible Spectrophotometer, absorbance was continuously monitored at 405 nm. The PON1 unit was defined as the enzyme quantity that disintegrates 1 μmol paraoxon substrate in one minute (Eckerson et al., 1983).

Purification of paraoxonase-1 (PON1) from amniotic fluid
The method given here has yielded an enzyme preparation acceptable for amniotic fluid. All steps were performed at 4 o C unless otherwise stated.
Step I: Collection of amniotic fluid Pregnancies at last week of gestation were pooled, which were planned from amniocentesis to evaluate other gestational diseases. The specimens were obtained from the Al-Betool hospital, Mosul/ Iraq. A 23-G spinal needle was used to aspirate the amniotic fluid from the abdomen by a medical professional specialized in the field of prenatal diagnostic procedures and fetal medicine. Samples (21) were centrifuged at 20,000 g for 30 min, and the supernatant was used to isolate PON1.

Step III: Cooling ultracentrifuge separation
The suspension produced from step II was centrifuged at 9000 g for (45) min at -4C to avoid denaturation. The protein in precipitate and supernatant are determined using by the modified Lowry method (Schacterle and Pollack, 1973), PON1 activity was determined in each fraction (Eckerson et al., 1983).

Step IV: Dialysis
Dialysis was performed using a semi permeable cellophane dialysis membrane with M.wt. cut off (<10000) dalton. The dialysis sac containing the suspension in (Step III) was dialyzed against 0.1M ammonium bicarbonate, was stirred with a magnetic stirrer overnight at 4 o C. The solution of dialysis was changed three times (per 3 hours) during dialysis (Robyt and White, 1987). The protein of the dialyzed suspension was used to estimate enzyme activity by modified Lowry method (Schacterle and Pollack, 1973).

p-nitrophenol Paraoxon Diethyl phosphate
Step V: Freeze-dryer (Lyophilization) technique The protein of the dialyzed enzyme (Step IV) was concentrated using a freeze-dryer (Lyophilization).
Step VI: Gel filtration chromatography using Sephadex G-50 and Sephadex G-100 The sephadex gel G-50 and G-100 supplied as a powder were suspended in adequate amont of distilled water so that when it was stirred it allows the incorporated air bubbles to escape rapidly to the surface. It was then allowed to swell in water bath for 3 hours at 90 o C to complete swelling. This suspension was used for column packing and sample application (Robyt and White, 1987).
In the present study, the column used was 2.0  90 cm which contain gel sephadex at a height of (85) cm. The exclusion limit for sephadex G-50 is (30000) Dalton, while sephadex G-100 is (150000) (Robyt and White, 1987).
A concentrated sample (4) ml of the protein material (from Step V) was applied on the top of a bed sephadex G-50 and the results applied on the top of a bed sephadex G-100, followed by buffer solution (0.05 M Tris-HCl, pH 8.0, and 0.2 M NaCl).
Elution of the protein materials was carried out at a flow rate for sephadex G-50 column at (75)ml/ hour with a definite time (4) min, but for sephadex G-50 column a flow rate (62)ml/ hour with a definite time (5) min. using buffer solution (0.05 M Tris-HCl, pH 8.0, and 0.2 M NaCl) as eluant. The fractions were collected using a fraction collector. The protein compounds in each fraction collected were detected by the absorbance at wave length (280) nm using UV/Visible Spectrophotometer. Peaks were detected from the plot of absorbance versus elution volumes. PON1 was determined in each fraction (Burtis et al., 2012).

Step VII: Freeze-dryer (Lyophilization) technique
The enzyme fraction from step (VI) which was obtained from gel filtration was dried using a freeze-dryer (Lyophilization) technique to obtain a powder or a concentrated protein. The enzyme was kept in a deep freeze at -20 o C in a tight sample tube to be used in further investigations.
Step VIII: Electrophoresis Samples from step VII which were applied on Sodium Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis (SDS-PAGE) using slab electrophoresis unit quick fit instrumentation (Laemmli, 1970).

Animals and Experimental protocol
Thirty five white Albino male rats, weighing 200-300 gm were used in this study; they were obtained from and maintained in the animal house of the Veterinary College, University of Mosul under conditions of controlled temperature. Rodent food rich in nutrient and tap water were used as bedding. The animals were divided into five groups of seven animals each and treated as follow: Group I-received single intraperitoneal (IP) dose of normal saline, this group served as a negative control. Group II -received single IP dose of cisplatin (10mg/kg), this group served as positive control. Group III-pretreated with IP dose of PON1 isolated alone (1mg/kg/day). Group IV and V-animals pretreated with IP dose of isolated PON1 (0.5mg and 1mg /kg/day respectively) for 5 successive days before and 5 successive days after single IP cisplatin (10mg/kg). Animals were anesthetized by ether, blood was collected directly by orbital sinus puncture (Intraocularly) and poured into plain tubes (Atta et al., 1983), the clot was dispersed with glass rod and then centrifuged at 3000 X g for 15 minute ; the serum was used within 2 days for the estimation of alkaline phosphatase (ALP) (Kind and King, 1954), aspartate aminotransferase (AST) (Reitman and Frankel, 1975), alanine aminotransferase (ALT) (Reitman and Frankel, 1975), creatine kinase (CK) (Szasz et al., 1976), total bilirubin(TB) (Martinek, 1966) and malondialdehyde (MDA) (Muslih et al., 2002). The data presented as Mean + SD. The significance of the differences between the mean values were calculated using unpaired student's t-test. p-values equal or less than 0.05 were considered to be significantly different (Indrayan and Sarmukaddam, 2001).

RESULTS AND DISCUSSION Enzyme purification
The results predicted that the enzyme activity was found in the (67)% of a saturation ammonium sulfate precipitates. As shown in Table (1), the specific activity was increased after dialysis. This might be due to the removal of the small molecules (Below 14000 Dalton) and increasing the purification of PON1.

Gel filtration separations
This technique was applied to separate the protein as a source of enzyme, which was obtained after dialysis and using a column containing sephadex G-50 and G-100 gel as shown in (step V). The result (Fig. 1) indicated that there is mainly one peak for PON1in sephadex G-50 and after lypholization injected to G-100, was mainly one peak (A) (Fig. 2). The elution volume of peak (A) was (180.4) ml. The specific activity of the enzyme peak (A) was (4.809 U/mg protein) with 147 folds of purification compared to initial extract Table (1). In other studied, established that two allozymic forms of esterases account for the paraoxonase activity present in human serum (Playfer et al., 1976;Smolen et al., 1991, Huang et al., 1995.

Molecular weight determination of PON1 by gel filtration
The molecular weight of peak (A) as a source of PON1 was determined by the gel filtration chromatography using sephadex G-100 column (2  85)cm calibrated with known molecular weight proteins that were listed in Table (2).  Table (2) gives a straight line as illustrated in Fig. (3). Log.

Fig. 3: A plot of the logarithm molecular weights of known proteins versus elution volume on a sephadex G-100.
The molecular weight of unknown protein compound separated by the column chromatography as shown in (step V) was determined from the standard curve, which was represented by Fig. (3). The comparative molecular weight of peak (A) as a source of PON1 is approximately equal to (43873 + 350) Dalton.

Molecular Weight Determination by SDS-PAGE
The electrophoretic mobility of PON1 using SDS-PAGE. The enzyme migrated as a single band in amniotic fluid only as shown in Fig. (4) with an apparent molecular weight of (43682+ 278) Dalton which was determined using known molecular weight compounds as shown in Fig. (5).  Rodrigo et al., 1997.

Optimum conditions for PON1 activity
To develop assay conditions where PON1 from amniotic fluid shows a maximum activity, a series of experiments were performed. These included enzyme concentration, pH of the assay conditions, incubation time, incubation temperature and substrate concentration (Murray et al., 2009).

Effect of enzyme concentration on PON1 activity
It is important to establish that the activity varies linearly and decreased finally with enzyme concentration. The activity of the enzyme was measured in the presence of different concentrations of partially purified enzyme from AF between (10-100) µg/ml as shown in Fig. (6).

Fig. 6: Effect of different protein concentrations on PON1 activity
The result indicated that the enzyme activity increased by increasing the concentration of protein as a source of the enzyme. These result agree with other research (Cunha Bastos et al., 1999) using isolated PON1 from liver of Pacu, Piaractus Mesopotamicus Holmberg. For the next experiment (80)µg/ml, as a source of the enzyme was selected for determination other optimum conditions.

Effect of Buffer Solution 2.1. Effect of buffer concentration on PON1 activity:
The activity of enzyme was measured in the presence of different concentrations of buffer solution within the range (0.06-0.18) mol/liter of Tris-HCl buffer at pH 8.5. Maximum activity was obtained using (0.14) mol/liter of Tris-HCl buffer (Fig. 7).

Effect of pH on the PON1 activity:
The influence of pH on the activity of PON1 was investigated using (80 µg/ml) as a source for enzyme from AF in (0.14) mol/liter Tris-HCl buffer. The assay conditions were conducted in the same manner as described earlier at pH range of (5.5 -10.0). Maximum PON1 activity was obtained at pH (8.0) as indicated in (Fig. 8). Extremes of pH above of 8.0 can lead to denaturation of the enzyme and decreased activity, because the structure of the catalytically active site for the enzyme depends on the ionic character of the amino acid side chains (Harvey and Ferrier, 2014). In other studies, the optimum pH of the enzyme was about (8.5) from rat liver (Rodrigo et al., 1997) and from the liver of Pacu, Piaractus Mesopotamicus Holmberg (Cunha Bastos et al., 1999).

Incubation time as a function of enzyme activity
To determine the stability of PON1 activity under assay conditions, a series of experiments were performed at different time intervals. The results indicated that maximum enzyme activity was obtained after (7) min. in (37 °C) incubation (Fig. 9).

Effect of temperature on PON1 activity:
It has been found that as the temperature increased, there was a concave up increase in the enzyme activity until it reached a maximum value at a temperature of (45 °C) then dropped gradually after that (Fig. 10). This increase is the result of the increased number of molecules having sufficient energy to pass over the energy barrier and form the products of the reaction. Further elevation of the temperature results in a decrease in reaction velocity as a result of temperature-induced denaturation of the enzyme (Harvey and Ferrier, 2014). Rat liver paraoxonase appeared in Rodrigo et al., 1997 relatively stable at 47.5 °C, with 50% of its activity remaining after 30 min of incubation. However, at 52.5 and 55 °C, rapid inactivation occurred after 15 and 60 min respectively.

Effect of substrate concentration on the enzyme activity:
To determine the effect of substrate concentration [Paraoxon] on the enzyme activity, a series of experiments were performed where the concentration of the substrate was varied (Fig. 11).  (12) giving a K m value of (3.79 mmol/liter) and V max (80.0 µmol/min.). Other report fund the K m for liver paraoxonase as 1.69 mmol/liter (Rodrigo et al., 1997). Karacabey Merino reported 49x10 -6 mmol/liter and 122 U/ mL for K m and V max values respectively (Arslan et al., 2011).

Optimum conditions of the purified paraoxonase-1:
The optimum conditions of the purified Paraoxonase-1 from amniotic fluid are shown in the following Table (3):

The effect of paraoxonase-1 isolate against cisplatin-induced hepatotoxicity and cardiotoxicity:
The results and the effect PON1 isolation on the serum levels of ALP, ALT, AST, CK, TB and MDA for the treated animals are listed in Table ( The results indicated that the serum levels of ALP, ALT, AST and MDA were significantly elevated in the group II in comparison with the group I (p<0.05). While in group IV and group V the serum levels of ALP, ALT, AST and MDA were significantly reduced. The cytotoxic effect of cisplatin is enhanced by the elevation of the dose, however, at higher doses, the less common toxic effects, such as hepatotoxicity and cardiotoxicity, may arise (Santos et al., 2007). It has been suggested that oxidative stress is an important mechanism of cisplatin-induced toxicity possibly due to depletion of reduced glutathione (GSH) (Yilmaz et al., 2004) The oxidative stress through the generation of reactive oxygen species, decreases antioxidant defense system including antioxidant enzymes and non enzymatic molecules (GSH) which are the major alterations in the cisplatin toxicity (Kart et al., 2010;Kocik et al., 2012). Also many studies reported that there were a significant elevation in the hepatic malondialdehyde (MDA) and reduction in the level of antioxidant enzymes in rats treated with cisplatin (Yilmaz et al., 2005;Mansour et al., 2006). Transaminases for (ALT and AST) as well as, ALP are the most sensitive biomarkers directly implicated in the extent of cellular damage and toxicity because they are cytoplasmic in location and are released into the circulation after cellular damage (Stockham and Scott, 2002). Elevation of the serum levels of the hepatic enzymes and bilirubin are the indicators for impaired liver functions (Iseri et al., 2007) (Table 4, group II). Cisplatin elevates serum cardiotoxicity enzymatic indices (AST and CK) and causes severe histopathological lesions in cardiac tissues . The effect could be a secondary event following cisplatininduced lipid peroxidation of cardiac membranes with the consequent increase in the leakage of AST and CK from cardiac myocytes . There are many evidence dealing with the administration of antioxidants that may be effective in ameliorating cisplatin-induced cardiotoxicity, like acetyl-L-carnitine, DL-α-lipoic acid and silymarin, which have been proven to possess antioxidant potentials and appear to be potential candidates to ameliorate cardiotoxicity associated with cisplatin use in rats (Mansour et al., 2008).
The improvement of liver and cardiac markers when animals pretreated with IP dose of isolated PON1 (0.5mg and 1mg /kg/day respectively) for 5 successive days before and 5 successive days after single IP cisplatin (10mg/kg) ( Table 4, Group IV and V) is due to contribute in of PON1 80 0.14 8.0 7 45 12 3.79 80.0 by removing toxic compounds and inhibit lipid oxidation in low density lipoproteins (LDLs) (Povey, 2010). Thus, it might be an important detoxifying enzyme and may play a protective role against the development of various diseases. Therefore PON1 could be defined as an antioxidant enzyme (Deakin and James, 2004;Ekinci and Beydemir, 2009). It is able to inhibit coppercatalyzed LDL oxidation in vitro and the inflammatory response induced by oxidized LDL in the arterial wall (Harangi, 2002). The second important function of PON1 is its anti-atherogenic activity. Serum PON1 exists with HDL in plasma and acts to prevent the oxidation of plasma lipoproteins (Aviram, 1999). PON1 is effective on lipid peroxides and also on hydrogen peroxide therefore, it is considered to have peroxidase like activity (Memişoğullar and Orhan, 2010). Serum PON1 activity is especially important for the protection of LDL phospholipids against oxidation. In the protective effect against atherosclerosis, LDL has different antiatherogenic features such as protection from free radical induced oxidation of LDL cholesterol in artery wall (Aviram, 1999). In addition to LDL, PON1 protects HDL too which is a carrier of lipid peroxide (Azarsiz and S ِzmen, 2000). PON1 protects lipoproteins and arterial cells against oxidation, probably by hydrolyzing lipid peroxides such as specific oxidized cholesteryl esters and phospholipids. It was suggested to contribute to the antioxidant protection conferred by HDL on LDL oxidation (Mackness et al., 1991).