Drug and xenobiotic metabolising enzymes in camel liver: Multiple forms and species specific expression

Camp. Eiochem. Physiol. Vol. 104C, No. I, pp. 137-145, 1993 Printed in Great Britain

03064492/93 $6.00 + 0.00 0 1993 Pergamon Press Lid

DRUG AND XENOBIOTIC METABOLISING ENZYMES IN CAMEL LIVER: MULTIPLE FORMS AND SPECIES

SPECIFIC EXPRESSION

H. RAZA and W. MONTAGUE

Department of Biochemistry, Faculty of Medicine and Health Sciences, U.A.E. University, P.O. Box 17666, Al Ain, United Arab Emirates (Fax 638-247)

(Received 22 June 1992; accepted for publication 3 1 July 1992)

Abstract-I. Previous studies have demonstrated the presence of phase I mixed-function oxidases (cytochrome P,,,-dependent) and phase II conjugation (glutathione S-transferase) enzymes in camel liver. This study represents further characterisation of these drug metabolising enzyme systems in camel liver by comparing their catalytic and immunochemical properties with enzymes of rat and mouse liver.

2. Usmg the specific PdsO substrate aniline, the microsomal aniline hydroxylase activity of camel liver was found to be significantly lower than that of rat and mouse. The K,,, values of the enzyme for aniline was similar in rat and camel liver; however, the V,,,,, for camel liver enzyme was 50% of the rat liver enzyme. Aminopyrene N-demethylase activity in camel liver, was lower than that of rat but higher than in mouse. Microsomal NADPH cytochrome C-reductase and NADPH-supported lipid peroxidation activities were similar in all three species.

3. The cytosolic phase II conjugation enzyme glutathione S-transferase and glutathione peroxidase activities in camel liver were markedly lower than those of rat and mouse enzymes. However, GSH concentration was similar in all three species.

4. Immunodot blot and Western blot analysis of liver cytosols, using antibodies to specific GST isoenzymes, have shown that camel liver like mouse and rat, expresses predominantly the Alpha and Mu classes of GST. GST Pi on the other hand, was abundant in mouse liver and was underexpressed in camel and rat liver.

5. Our results demonstrate that there are multiple forms of phase I (P,& and phase II (GST) enzymes in camel liver and that they are comparable with the drug metabolising enzymes of rat and mouse. The lower a&ties of drug metabolising enzymes in camel liver compared with rat and mouse appear to be related to the differential expression of selective Pd5,, and GST isoenzymes.

INTRODUCllON

Hepatic cytochrome Pd5,, (PdsO) and glutathione S-transferase (GST) are the most active drug metabolising enzymes that participate in the metabolism of a wide variety of exogenous and endogenous compounds in mammals (Jakoby, 1977; Gonzalez, 1989; Guengerich, 1991). These compounds include drugs, chemical carcinogens, pollutants, fatty acids, steroids, vitamins, prostaglandins and leukotrienes (Chasseaud, 1979; Guengerich et al., 1982; Nebert and Gonzalez, 1987; Chang et al., 1987). Many isoenzymes of Pbso have been purified from mammalian tissues and characterized on the basis of their catalytic and molecular properties (Guengerich et al., 1982; Nebert and Gonzalez, 1987; Raza and Avadhani, 1988; Gonzalez, 1989; Raza et al., 1992a). Similarly several GST isoenzymes have been ident- ified in mammalian tissues (Mannervik et al., 1985; Hayes and Mantle, 1986; Raza et al., 1991). Man-

List of abbreviations: GSH, reduced glutathione; Pd5,,, cytochrome Peso, GST, glutathione S-transferase; GSH-Px, glutathione peroxidase; CDNB, I-chloro2-4-dinitrobenzene; NADPH, reduced nicotinamide adenine dinucle- otide phosphate; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis.

nervik et al. (1985) have characterised mammalian GST isoenzymes and classified them into three distinct classes: Alpha (basic), Mu (neutral) and Pi (acidic). All three isoenzymes are immunochemically distinguishable and exhibit substrate specificity (Mannervik et al., 1985 and Raza et al., 1991).

The major role of drug metabolising enzymes is to convert lipid-soluble compounds into water-soluble metabolites with diminished biological activity. How- ever, in some instances, these enzymatic changes may generate chemically reactive metabolites which can activate physiological or pathological processes (Conney, 1982; Guengerich, 1991). The balance of metabolic activation and inactivation of drugs and other xenobiotics in particular tissues is an important factor in organ-specific toxicity. Although PdsO is normally considered as an activation enzyme, and GST as a detoxification enzyme (Gibson and Skett, 1986), the relative expression of the various isoenzymes of P,,, and GST is critical in determining the balance between toxication and detoxication path- ways.

The GST isoenzymes conjugate reactive electrophilic compounds with cellular nucleophilic glutathione (GSH) and play an important role in promoting cellular resistance to chemotherapeutic

137

138 H. RAZA and W. MONTAGUE

drugs and preventing toxic injuries in a variety of tissues (Chasseaud, 1979; Ketterer, 1988; Sato, 1989). An overexpression of GST isoenzymes in the early stages of some cancers is considered to be a marker for preneoplasia (Sato, 1989). Intracellular GSH, in addition to its role in the conjugation of electrophilic compounds, also acts as an antioxidant to prevent the toxic injuries caused by oxidative stress and membrane lipid peroxidation during the metabolism of many endogenous and exogenous compounds (Jakoby, 1977; Levine, 1982; Raza and Levine 1986; Bast and Haenen, 1988).

Despite an enormous amount of information on P,,, and GST in a variety of mammalian species very little is known of the drug metabolising properties of camel liver. The existence of phase I mixed-function oxidases (PdsO dependent) and phase II (GST) dependent drug metabolism in liver and extrahepatic tissues of the camel was reported recently (El Sheikh et al., 1988; El Sheikh et al., 1991). However, the properties of enzyme systems, their substrate specificities, isoenzyme forms and the regulation of their activities have not been defined. In this study we have investigated the general properties of the drug metabolising systems in subcellular fractions from camel liver. Our results suggest the existence of multiple forms of P,,, and GST in camel liver which are comparable to those of rat and mouse enzymes.

MATERIALS AND METHODS

Materials and supplies

I-Chloro-2,4_dinitrobenzene (CDNB), ethacrynic acid, 4-aminophenol, aniline-HCl and p -aminophe- no1 were obtained from Aldrich Chemical Co. (Mil- wauki, WI, U.S.A.). NADPH, cytochrome C, reduce glutathione (GSH), thiobarbituric acid (TBA), dithiobisnitrobenzoic acid (DTNB), and Mg2+-ADP were purchased from Sigma Chemical Co. (St Louis, MO, U.S.A.). Chemicals, reagent kits and materials for SDS-PAGE and for immunoblotting (Dot and Western blot) and for colour development were purchased from Bio-Rad Laboratories (Richmond, CA, U.S.A.). All other chemicals used were purchased from Sigma (U.S.A.), Aldrich (U.S.A.) and Fisons (U.K.).

Purified GST isoenzyme and antibodies

GST isoenzyme Pi was purified from skin by affinity chromatography as described by Raza et al. (1992b). SDS-PAGE analysis of purified GST Pi indicated a single major band migrating as 22.5 kDa protein (Fig. 3, lane GST Pi). This preparation was used as the reference in this study.

Polyclonal antibodies to purified Alpha, Mu and Pi classes of human GST were a generous gift from Professor Y. C. Awasthi, University of Texas Medi- cal Branch, Galveston, Texas, U.S.A. These antibodies appear to crossreact monospecifically with rodent and human GSTs (Raza et al., 1991).

Animals and isolation of subcellular fractions

Liver tissues from three adult male camels (Camelus dromedarius) were obtained from a local slaughter house. Liver from adult male Sprague-Dawley (200-250 g) rats and from Swiss albino mice (25-35 g) were obtained from the U.A.E. University animal house facility. Livers from all three species were immediately washed with ice cold saline (0.9% NaCl) and 1 g pieces were each homogenised in 4.0 ml of 10 mM potassium phosphate buffer, pH 7.4, containing 0.15 M KCI. Microsomes and post microsomal (cytosolic) fractions were prepared by ultracentrifugation as described by Raza and Levine (1987). Protein concentration was measured by the method of Bradford (1976) using bovine serum albumin as a standard.

Microsomal mixed-function oxidase (Pd5,,) system

Microsomal P.,50 dependent mixed-function oxidase activities in camel, rat and mouse liver were measured using aminopyrene and aniline as the substrates which are believed to be metabolised by distinct isoenzymes of P,,,. Aminopyrene-N-demethylase activity was determined according to the method of Cochin and Axelrod (1959). Aniline hydroxylase activity was measured by the method of Guarino et al. (1969). NADPH-cytochrome C reductase activity was determined as described by Dignam and Strobe1 (1977).

Microsomal lipid peroxidation

NADPH dependent microsomal lipid peroxidation was measured using a modification of the procedure described by Dixit et al. (1983). This is based on determination of the end product of lipid peroxidation, malonedialdehyde, as estimated by thiobarbituric acid (TBA) test. The complete assay system in I .O ml of 10 mM potassium phosphate buffer, pH 7.4, contained 1 mM NADPH, 1 mM MgCI,, 1 mM FeSO,, 0.1 mM ADP and 2-3 mg of microsomal proteins. After 30min incubation at 37°C the reaction was stopped by adding 2.0 ml of 15% TCA containing 0.375% TBA and 0.25 N HCI. Finally the tubes were heated at 90°C for 15 min and centrifuged to remove any precipitate. The malonedialdehyde formation in the supernatant was measured spectrophotometrically at 532 nm. Activity was calculated using a molar extinction coefficient of I .56 x IO’. Controls without NADPH and containing boiled microsomes were also run in parallel.

Cytosolic GSH-dependent enzymes

GST activities with I-chloro-2,4_dinitrobenzene (CDNB) and ethacrynic acid as substrates were measured in the cytosolic fraction of camel, rat and mouse liver according to the method of Habig et al. (1974) as described previously (Raza et al., 1991). Selenium-dependent GSH-peroxidase (GSH-Px) activity was monitored spectrophotometrically in a reaction which couples the reduction of H?O? by

Drug metabolising enzymes in camel liver

Table 1. Mixed-function oxidase activitia and lipid peroxidation in hepatic microsomcs prepared from camel, rat and mouse

Enzvme activities* Camel Rat Mouse

Aminopyrene-N-demethylase 4.28 f 0.297 5.85 k 0.33 3.38 k 0.16

139

(Formaldehyde formed) Aniline hydroxylase 0.45 f 0.07t 1.47 f 0.05 1.73*0.11

(p-aminophenol formed) NADPH-cytochrome C-reductase 265.19 + 12.27 275.20 & 11.67 279.57 + 11.70

(cyt. C reduced) Lipid peroxidation 0.39 f 0.03 0.42 f 0.05 0.37 f 0.04

(malonedialdehyde formed)

Enzyme activities and rate of lipid peroxidation were measured as described in Materials and Methods. The values shown are mean f SEM of three different experiments run in duplicate.

*nmol product formed/min/mg of microsomal protein. tsignificantly different (P i 0.05) from rat and mouse liver microsomal enzyme

GSH to the oxidation of NADPH with GSH-reductase according to the method of Pagila and Valen- tine (1967).

Cytosolic GSH concentration was measured by the method of Buttar et al. (1977).

Polyacrylamide gel electrophoresis and Immunoblot analysis of proteins

Cytosolic proteins from camel, rat and mouse liver and purified GST Pi from rat skin were separated on SDS-PAGE according to the method of Laemmli (1970) using a 12% slab gel. The resolved bands were visualised by staining with Coomassie Blue as described previously (Raza et al., 1991).

Proteins resolved by SDS-PAGE were electrophor- etically transferred onto nitrocellulose membranes by Western blotting as described by Towbin et al. (1979). Specific antibody interaction with various GST isoenzymes was determined by incubating the membrane with polyclonal antibodies as described before (Raza et al., 1991).

In some cases the cytosolic fractions from camel, rat and mouse liver were applied directly to nitrocellulose membranes (0.45 ,um pore size) using Dot Blot (Bio-Rad, U.S.A.) apparatus. Specific antibody reactivity with cytosolic proteins was determined using alkaline phosphatase conjugated anti-rabbit secondary antibody. The presence of bound secondary

l/Km pM Aniline - HCI l/S

Fig. 1. Kinetics of aniline hydroxylase activity. Microsomal preparations (1.5 mg protein) from camel and rat liver were used to determine the K, and V,,,,, values of aniline hydroxylase activity under optimal reaction conditions as described in “Materials and Methods”. The graph represents the double reciprocal

plot of Lineweaver-Burk.


NADPH dependentmte of microsomal lipid peroxldation In camel liver

20-

Tint@ (min)

Fig. 2. Rate of lipid peroxidation. NADPH dependent lipid peroxidation in camel liver microsomes was measured at different time intervals as described in “Materials and Methods”.

antibody was detected as described by Raza and Avadhani (1988).

RESULTS

Microsomal drug metabolising enzymes

Table 1 shows the microsomal drug metabolising enzyme activities in camel, rat and mouse liver. The aminopyrene-N-demethylase activity of camel liver was 4.28 + 0.29 nmol/min/mg protein compared to 5.8 + 0.33 and 3.4 f 0.16 nmol/min/mg for rat and mouse. In contrast, aniline hydroxylase activity in camel liver was only 33% of the rat and 25% of the mouse enzyme activity. Kinetics analysis of camel liver aniline hydroxylase by Lineweaver-Burk plot (Fig. 1) showed that the camel liver enzyme had an apparent K,,, of 28 PM and a V,,,,, of 1.1 nmol/min/mg protein, while the rat enzyme had a K,,, of 25 PM and a V,,, 2.0 nmol/min/mg protein. The results in Table 1 show that the enzyme activities in camel liver are significantly lower than that in rat liver. However, while the aniline hydroxylase activity

Table 2. Effect of NADPH and Fe SO, on lipid peroxidation activity of camel liver microsome

Additions or Malonedialdehyde formed deletions nmol/min/mg protein

Complete 0.376 (100%) -NADPH 0.066 (17.5%) -Fe SO, 0.053 (14%) Boiled microsome 0.163 (43%)

Complete assay system contained, in I ml of IO mM KPi buffer pH 7.4, I mM NADPH, I mM MgCI,, I mM Fe SO,, 0.1 mM ADP and 2 mg microsomal protein from camel liver. Malonedialdehyde formation was estimated as described in Materials and Methods. Values in parenthesis are the percentage of activity compared to the complete assay system.

in camel liver is significantly lower than that of mouse liver the aminopyrene N-demethylase activity is slightly higher. The activity of NADPH-cytochrome C (P,,O) reductase, another component of microsomal mixed-function oxidase system which transports elec- tron from NADPH to P4=,,, in vioo, is similar in all three species (Table 1).

The NADPH-dependent microsomal lipid peroxidation was not significantly different in camel, rat or mouse liver (Table 1). Lipid peroxidation partially appears to be an NADPH-dependent enzymatic reaction (Fig. 2 and Table 2) and requires Fe*+ ions since the omission of NADPH or Fe*+ ions and using boiled microsomes inhibited the process (Table 2).

Cytosolic enzymes

Cytosolic GST and GSH-Px activities of camel liver are different to those of rat and mouse liver (Table 3). GST activity of camel liver with CDNB as

Table 3. Glutathione concentration, GST and GSH peroxidase (GSH-Px) activities in hepatic cytosol from camel, rat and mouse

Assays

GSH

(mM)

Camel Rat

2.97 f 0.28 3.46 + 0.25

Mouse

2.67 + 0.19

GST 0.77 f 0.035 I .32 k 0.05 I.91 +0.04 (CDNB)’

GST 28.54 + I .4$ 71.03 + 3.25 71.16k3.47 (ethacrynic-acid)t

GSH-Pxt 35.87 f 3.05 604.75 + 5.21 791.75 + 24.7

GSH concentration, GSH-dependent GST activities with CDNB and ethacrynic acid as substrates and GSH-PX activity in camel, rat and mouse liver cytosol. were determined as described in Ma- terials and Methods. Values are the mean f SEM of three determinations.

*pmol conjugated/min/mg cystosolic protein. tnmol conjugated/min/mg cytosolic protein. fnmol NADPH oxidized/min/mg cytosolic protein. §Significantly different (P < 0.05) from rat and mouse liver cytosohc

enzyme activities.

Drug metabolising enzymes in camel liver 141

a substrate was 59% and 40% of the activity in rat and mouse liver respectively. Similarly, activity with ethacrynic acid was also about 40% of that of rat and mouse liver. GSH-Px activity in camel liver was also markedly lower (Table 3) than that of rat and mouse liver (16 and 22 fold lower respectively). As shown in Table 3 the lower activities of GSH dependent enzymes in camel liver are not due to the low concentration of GSH since no significant difference was observed in hepatic GSH concentration in all three species.

Characterisation of camel liver GST

The electrophoretic separation of cytosolic proteins from camel, rat and mouse liver is shown in Fig. 3. Purified GST Pi migrates at a position corre- sponding to an apparent molecular mass of 22.5 kDa.

Western blot analysis of the separated cytosohc proteins and purified rat skin GST Pi revealed that only the purified enzyme and a 22.5 kDa protein in mouse cytosol, comigrating with the purified enzyme, exhibited significant immunocrossreactivity with

antibodies to GST Pi (Fig. 4). Rat and camel cytosols showed very poor reactivity. Purified GST Pi, as expected, did not crossreact with the antibodies to GST Mu and Alpha class of enzymes. However, antibodies to GST Mu and Alpha classes of isoenzymes showed strong immunocrossreactivity with the cytosols from all three species in the region of 2428 kDa (Fig. 4).

The results also demonstrate that the subunit com- positions of GST Mu and Alpha and their relative expression in all three species appears to be different. The GST Mu antibody crossreacted with a 25.5 kDa protein in mouse, with a 26.5 kDa protein in camel and with a 26 kDa protein in rat liver (Fig. 4). GST Alpha antibody recognized three protein bands (molecular mass 28.5 kDa, 27 kDa and 24.5 kDa) in rat cytosol (Fig. 4) while in mouse and camel liver the antibody recognises dimers of GST (molecular mass 24.5 kDa and 27 kDa). As shown in the Western blot the camel liver showed relatively lower amounts of GST protein when the same amount of rat and mouse cytosolic proteins were resolved by electrophoresis.

KDa

100

75

32

27

Fig. 3. SDS-PAGE analysis of cytosolic proteins and purified GST Pi. Cytosol prepared from rat, mouse and camel liver (5Opg protein) were subjected to 12% SDS-PAGE as described in “Materials and Methods”. Purified GST Pi from rat skin showed a migration at 22.5 kDa (indicated by an arrow) molecular mass. Gel was stained by Coomassie Blue. Molecular weight is molecular weight markers

in kDa.


Fig. 4. Western blot analysis of rat, mouse and camel liver cytosol. Cytosol(50 pg protein) from rat, mouse and camel (three animals) liver were subjected to 12% SDS-PAGE as described in Fig. 3. The proteins were transferred onto nitrocellulose membrane according to Towbin er al. (1979) and probed with the

antibodies to GST Alpha, Mu and Pi as described by Raza er al. (1991).

This also supports the observation of lower GST activity in camel liver.

Immunodot blot analysis of liver cytosolic proteins (Fig. 5) confirmed the predominant expression of Alpha and Mu classes of GST in all three species. While GST Pi was expressed in mouse liver but not in camel and rat.

DISCUSSION

The drug metabolising ability of camel liver was investigated recently by El Sheikh et al. (1988 and 1991) but the studies used a limited number of substrates which did not allow determination of the normal spectrum of constitutive drug metabolising enzymes. Many drug metabolising enzymes are poorly expressed in normal liver but are markedly induced after the treatment with various drugs and carcinogens (Nebert and Gelboin, 1969; Guengerich, 1982; Raza and Avadhani, 1988; Raza et al., 1992a). Furthermore, the earlier studies on camel liver were conducted using crude preparations of either liver homogenate or 10,000 g supernatant. We therefore studied the drug metabolising enzymes in subcellular fractions from normal camel liver using various substrates and have demonstrated the existence of multiple forms of enzymes which exhibited unique substrate specificity and immuno-cross-reactivity. Our results are in general agreement with the earlier reports which indicate reduced ability of camel liver

to metabolise foreign chemicals and drugs when compared to other species (Ali and Hassan, 1986; El Sheikh et al., 1988). In addition we have demonstrated (Tables 1 and 3) that the activities of both microsomal phase I enzymes of activation and cytosolic phase II enzymes of detoxication were lower in camel liver when compared to those of rat and mouse liver. Furthermore both phase I and phase II enzymes in camel liver exhibit substrate specificities which suggest the presence of multiple forms of enzyme which are comparable with the isoenzymes from rat and mouse liver (Tables 1 and 3). This difference between camel and rat and mouse was seen in selective microsomal Pd5,, and cytosolic GSH dependent enzymes but not in the microsomal NADPH-cytochrome C (P,,,,) reductase activity, lipid peroxidation and GSH concentration (Table 1 and 3). Levine (1982) and others (Das et ul., 1985; Raza and Levine, 1986; Bast and Haenen 1988) have shown that the GSH concentration and lipid peroxidation regulates the drug metabolising activities in the liver. An increase in microsomal lipid peroxidation and a decrease in cytosolic GSH concentration are thought to be the important factors in reducing the drug- metabolising enzyme activities. However, the lower drug metabolising activities of camel liver observed in the present study do not appear to be due to these factors, as the GSH concentrations and the rate of lipid peroxidation were similar in all three species.


Fig. 5. Immunodot blot analysis of rat, mouse and camel liver cytosol. Cytosol (20 ng protein) from rat, mouse and camel liver and putrified GST Pi from rat skin (2 pg protein) were directly applied onto nitrocellulose membrane using Bio-Rad Dot apparatus. lmmunoreactivities of cytosolic proteins with

GST antibodies were determined as described in “Materials and Methods”.

We have studied the multiplicity of GST in camel liver cytosol by determining the expression of specific isoenzymes by Western and Dot blot analysis using polyclonal antibodies to specific isoenzymes. The results of immunocrossreactivity of camel, rat and mouse hepatic cytosols are presented in Figs 4 and 5. Both Immunodot blot analysis of total cytosolic proteins and Western blot analysis indicated the expression of multiple forms of GST proteins in camel liver which is comparable with rat and mouse liver enzymes. Our data indicated that, like in the rat and mouse, the camel liver has an abundant expression of Alpha and Mu classes of GST. GST Pi was highly expressed in mouse liver but not in rat and camel. In this study, a poor expression of GST Pi in adult rat liver confirms earlier studies (Hayes et al., 1987; and Sato, 1989). Like other mammalian species (Mannervik et al., 1985) the GST Pi isoenzymes in camel liver may be a homodimer since a single faint immunoreacting band (on overexposure of blots to colour development reagents) at molecular mass 22.5 kDa was observed comigrating with pure GST Pi. Mouse GST Pi (molecular mass 22.5 kDa) also appears to be a homidimer (Fig. 4), confirming our earlier observations and observation made by others (Raza et al., 1991 and Mannervik et al., 1985).

The Alpha class of GST is the major isoenzyme expressed in camel liver (molecular mass 24.5 kDa and 27 kDa). This observation is in agreement with the earlier reports by Hunaiti and Sarhan (1987) who

reported seven different forms of basic GST isoenzymes (presumably representing Alpha class, as characterised by Mannervik et al., 1985) in camel liver cytosol. In rat cytosol, GST Alpha antibody cross-reacted with three proteins of molecular mass 24.5 kDa, 27 kDa and 28.5 kDa. Bass et al. (1977) have also reported that rat liver basic GST isoenzymes are heterotrimers.

GST Mu antibody recognises a 26.5 kDa major protein in camel liver (Fig. 4). These findings strongly indicate the existence of multiple isoenzymes of GST in camel liver which are comparable with other species.

In summary, our results demonstrated the occur- rence of multiple isoenzymes of GST in camel liver and suggest the possible existence of multiple isoenzymes of P,,, We have also provided evidence for the expression of specific drug metabolising enzymes in camel liver and suggested that the lower ability of camel liver to metabolise foreign chemicals and drugs may be related to the reduced expression of selective isoenzymes. An interesting and important area of future research will be to study the inducibility and substrate specificity of different forms of drug metabolising enzymes in camel liver under normal

and pathophysiological conditions,

AcknoM,ledgements-Authors thanks to Prof. Y. C. Awasthi for the generous gift of polyclonal antibodies used in this study. Thanks are also due to the technical as- sistance provided by Mr Mansoor Qureshi. We gratefully


acknowledge the help of Mr K. Assainar and the Media Production Unit of Faculty of Medicine and Health Sciences in preparing this manuscript.

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Drug and xenobiotic metabolising enzymes in camel liver: Multiple forms and species specific expression

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