Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology
Comparative liver metabolic enzyme activity of cytochrome P450 and glutathione-S-transferase in crocodile (Crocodylus siamensis) and livestock
Graphical abstract
Introduction
The freshwater crocodile natives in Thailand are the Siamese crocodile (Crocodylus siamensis). They are medium-sized, freshwater crocodilian, with a relatively broad, smooth snout, and an elevated, bony crest behind each eye. They feed mainly on fish and snakes, but also eat amphibians and small mammals. Siamese crocodile is an important economic animal in Thailand. The database from the Department of Fisheries Ministry of Agriculture and Cooperatives in 2017 indicated that two types of crocodiles are commercially raised in Thailand, they are the freshwater crocodile (Thai crocodile) and the saltwater crocodile. There are more than one thousand crocodile farms, raising about 1.2 million crocodiles in Thailand (BBC NEWS THAI, 2017).
There are several advantages from crocodiles; as previously reported by Pata et al. (2011), Siamese crocodile's white blood cells contain antimicrobial proteins and peptides, showing broad-spectrum antibacterial activity Phosri et al. (2014) reported that crocodile blood displays marked effects on cancer cells not only with anti-23 strains of bacteria but, including those resistant to antibiotics, also antioxidant and anti-inflammatory properties. Siddiqui et al. (2017) reported that crocodiles survive in polluted environments in the presence of endogenous antitumor compounds by the crude extracts of crocodile organs that contain active components that affect the viability of prostate cancer (PC3) cells. Sarhan and Al-Qahtani (2007) reported that the Dhub (Uromastyx aegyptia) liver has the highest antioxidant enzyme activity to counteract the oxidative damage. Alligator is a useful animal model for the assessment of wildlife exposure to environmental contaminants due to their potential for bioaccumulation as top predators and having a long life span, therefore hepatic biomarker is considered a suitable biomonitoring species for various toxins (Gunderson et al., 2016).
The liver is one of the largest organs in the crocodile body. It plays a significant role in metabolism and various functions in the body. These functions include glycogen storage, plasma protein synthesis, and detoxification, where the latter also plays a crucial role in biotransformation and detoxification of many endogenous and exogenous compounds. Two main types of metabolism deal with xenobiotics. The phase I detoxification system, composed mainly of the cytochrome P450 family enzymes which, are generally the first enzymatic defense against foreign compounds. As a consequence of this step-in, the detoxification process, reactive molecules, which may be more toxic than the parent molecule, are produced. If these reactive molecules are not further metabolized by phase II conjugation, they may cause damage to proteins, RNA, and DNA within the cell. Phase II conjugation reactions generally follow phase I activation, resulting in a xenobiotic that has been transformed into a water-soluble compound that can be excreted through urine or bile (Liska, 1998; Timbrell, 2008).
The phase I detoxification system is composed mainly of the cytochrome P450 (CYP) family of enzymes. The CYPs are a superfamily of enzymes containing iron heme. It is a microsomal monooxygenase, which requires NADPH as an external reductant. It is involved in the oxidative metabolism of many endogenous substances such as steroids and bile acids, as well as the detoxification of a wide variety of xenobiotics. The CYPs are a superfamily of enzymes (Ziglari and Allameh, 2013; Timbrell, 2008). CYP1A2 localizes to the endoplasmic reticulum and its expression is induced by some Polycyclic Aromatic Hydrocarbons (PAHs), some of which are found in cigarette smoke, and other xenobiotic substrates for this enzyme include caffeine, aflatoxin B1, and paracetamol (acetaminophen) (Zanger and Schwab, 2013). CYP2E1 is expressed mainly in the hepatocytes of the liver; however, significant amounts are also found in the Kupffer cells (Koop et al., 1991). CYP2E1 metabolizes and activates many toxicological substrates such as ethanol, carbon tetrachloride, acetaminophen, benzene, halothane, and many other halogenated substrates (Lu and Cederbaum, 2008).
The phase II detoxification system is composed mainly of glutathione-S-transferase (GST). GST catalyzes the conjugation of glutathione on the sulfur atom of cysteine to various electrophiles and catalyzes the conjugation of multiple electrophiles with glutathione and detoxifying both exogenously and endogenously derived toxic compounds (Cançado et al., 2005). GST is a large family of metabolic enzymes. Cytosolic GST in the alpha and pi classes are abundant in the hepatocytes; they are inducible by exogenous chemical substances and carcinogens like the aflatoxins in previous studies (Ziglari and Allameh, 2013).
The liver is the main organ that metabolizes xenobiotics. Aflatoxin B1 (AFB1) is metabolized in the liver through phase I (CYP450 isoforms), mainly by CYP1A2 and CYP3A4 isoenzymes, and converts to intermediate products of AFB1-exo-8, 9-epoxide (AFBO), AflatoxinB2a (AFB2a), Aflatoxin M1 (AFM1), AflatoxinQ1 (AFQ1), and AflatoxinP1 (AFP1). The AFBO metabolite form adducts with amino acids and DNA. The DNA adducts are moderately resistant to DNA repair processes, and this causes gene mutation and hence the development of cancers especially hepatocellular carcinomas. Phase II reactions are limited to the conjugation of the metabolite AFBO with glutathione to reduce the toxicity by GST (Ziglari and Allameh, 2013; Bbosa et al., 2013; Dohnal et al., 2014).
This study aims to compare the metabolism of liver cytochrome P450 enzymes activity focusing on CYP1A2 and CYP2E1 and GST enzyme activity for class pi, mu, alpha, and the universal GST between crocodile and livestock, plus, study the relationship of these two enzyme activities in crocodile.
Section snippets
Chemicals
Tris base (TRIS), potassium phosphate (monobasic), potassium phosphate (dibasic), sodium phosphate, hydrochloric (HCl), ethylenediaminetetraacetic acid (EDTA), sucrose, glutathione reduced form (GSH), 1-Chloro-2,4-dinitrobenzene (CDNB), trans4-Phenyl-3-buten-2-one (t-PBO), ethacrynic acid (EA), cumene hydroperoxide (CuOOH), magnesium chloride hexahydrate (MgCl2.6H2O), 3-Cyano-7-Ethoxycoumarin (CEC), 7-Methoxy-4(trifluoromethyl)-Coumarin (7-MFC), Glucose-6-phosphate (G-6-P) disodium salt,
Cytochrome P450 activities
The Michaelis-Menten equation plot of cytochrome P450 activity is shown in Fig. 1. The CYP2E1 was highest in pig > chicken > crocodile while CYP1A2 was highest in chicken > crocodile > pig at (p < 0.05) as shown in Fig. 1.
Glutathione-S-transferases activity
The Michaelis-Menten plot of cytocolic GST activity against the four kinds of substrate was done. The GST formations were determined by using a spectrophotometer according to (Habig et al., 1974) and the GST plot against each substrate is shown in Fig. 2. The level of km and
Discussion
The enzymatic activity of both CYP450 and GST was determined in the liver extraction from crocodile, similar to the findings in mammal and poultry. However, the GST enzyme activity of crocodile liver was higher than in pig and chicken. In contrast, the CYP450 enzyme kinetic activity towards CYP1A2 could not be calculated but CYP2E1 could be analyzed even though the amount of enzyme activity was low.
However, the determination of microsomal cytochrome P450 by using a spectrophotometer revealed
Conclusions
CYP1A1 and CYP2E1 existed in the freshwater crocodile livers microsomal enzyme activities and these enzymes could be determined in both pig and chickens. Conversely, the universal GST, alpha class, mu class and pi class of phase II existed in crocodile liver cytosolic fractions and these enzymes could also be determined in pig and chicken. The crocodile liver microsomal could metabolize AFB1 to AFM1, suggesting that CYP1A2 activity was presently active, as well as other CYP types, which must be
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
The Graduate Study Research Scholarship supported this study for International Publications, Kasetsart University Research and Development Institute (KURDI) and my appreciation for the sample supported by Punya Farm Kamphaengsaen Company.
References (36)
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal. Biochem.
(1976)- et al.
Human alpha class glutathione S-transferases: genetic polymorphism, expression, and susceptibility to disease
Methods Enzymol.
(2005) - et al.
Electrophoretic and immunological analysis of glutathione transferase isoenzymes of human kidney carcinoma
Biochem. Pharmacol.
(1989) - et al.
Glutathione-S-transferase activity toward aflatoxin epoxide in livers of mastomys and other rodents
Toxicon
(2002) - et al.
Variations in hepatic biomarkers in American alligators (Alligator mississippiensis) from three sites in Florida, USA
Chemosphere
(2016) - et al.
Glutathione S-transferases. The first enzymatic step in mercapturic acid formation
J. Biol. Chem.
(1974) - et al.
Role of CYP2E1 in the hepatotoxicity of acetaminophen
J. Biol. Chem.
(1996) - et al.
CYP2E1 and oxidative liver injury by alcohol
Free Radic. Biol. Med.
(2008) - et al.
Characterization of the novel antibacterial peptide Leucrocin from crocodile (Crocodylus siamensis) white blood cell extracts
Dev. Comp. Immunol.
(2011) Metabolism as a factor in determining the toxic action of the aflatoxins in different animal species
Food Cosmet. Toxicol.
(1973)