Metabolism of benzo(a)pyrene,dimethylbenzanthracene and aflatoxin B1 by camel liver microsomes

The ability of camel liver microsomes to metabolise a range of common environmental carcinogens including benzo(a)pyrene, dimethylbenzanthracene and aflatoxin B1 has been investigated. The camel liver has shown the ability to metabolise benzo(a)pyrene, dimethylbenzanthracene and aflatoxin B1 to a number of metabolites. The major metabolites of benzo(a)pyrene produced by camel liver enzymes were identified as its mono-hydroxy derivatives and suggest that the metabolic detoxification pathways of carcinogen metabolism are predominant in this species. Benzo(a)pyrene metabolising activity in camel liver required NADPH and was inhibited by CO and alpha-naphthoflavone suggesting the involvement of cytochrome P450 in the metabolism of this carcinogen by camel liver. The cytochrome P450-dependent metabolism of carcinogen and other specific substrates such as ethoxyresorufin and ethoxycoumarin, by camel liver enzymes, was about 50% higher than that of rat liver enzymes. The cytochrome P450-dependent metabolism of a variety of carcinogenic and other substrates by camel liver demonstrated that there are multiple forms of cytochrome P450 enzymes involved in the metabolism of a wide array of xenobiotics and pollutants.     

Molecular analysis of some camel cytochrome P450 enzymes reveals lower evolution and drug-binding properties

Camels bear unique genotypes and phenotypes for adaptation of their harsh environment. They have unique visual systems, sniffing, water metabolism, and heat-control mechanisms that are different from other creatures. The recent announcement for the complete sequence of camel genome will allow for the discovery of many secrets of camel life. In this context, the genetic bases of camel drug-metabolizing enzymes are still unknown. Furthermore, the genomic content of camel that rendered it highly susceptible to some drugs (as monensin and salinomycin) and became easily intoxicated needs to be investigated. The objectives of this work are the annotation of camel genome and retrieval of camel for cytochrome P450 (CYP) 1A1, 2C, and 3A enzymes. This is followed by comprehensive phylogenetic, evolution, molecular modeling, and docking studies. In comparison with the human enzymes, camel CYPs showed lower evolution rate, especially CYP1A1. Furthermore, the binding of monensin, salinomycin, alfa-naphthoflavone, felodepine, and ritonavir was weaker in camel enzymes. Interestingly, rerank score indicated instable binding of monensin and salinomycin with camel CYP1A1 as well as salinomycin with camel CYP2C. The results of this work suggest that camels are more susceptible to toxicity with compounds undergoing metabolic oxidation. This conclusion was based on lower evolution rate and lower binding potency of camels compared with the human enzymes.     

Molecular Characterization of the <Emphasis Type="Italic">Camelus dromedarius</Emphasis> Putative Cytochrome P450s Genes

The expression levels of cytochrome P450s were examined in different camel tissues by western blotting and semi-quantitative polymerase chain reaction. Camelus dromedarius liver microsomes were found to express different P450s isoenzymes constitutively. The maximum expression of P450 protein was seen in the camel liver in the order of P450 2E1, 1A1, 3A and 2B1/2. Camel extrahepatic tissues, kidney, spleen and the lung showed detectable levels of P450s 1A1 but lower than that noticed in liver. Detectable level of P450 2B1/2 was also observed in camel lung (29.5 vs. 58% liver microsomes). P450scc and 21-hydroxylase were found to be differentially expressed only in camel testis. Partial sequences of these P450s genes showed high similarities with the human P450s. These results demonstrate that the multiple forms of P450s are differentially expressed in camel tissues and that the relative levels of expression are comparable with other mammals. These observations might be important in understanding the differential susceptibility of camel tissues to the toxic effects of xenobiotics and environmental pollution.     

Camelus dromedarius Putative Cytochrome P450 Enzyme CYP2E1: Complete Coding Sequence and Phylogenetic Tree

This study determined the full-length sequence of CYP2E1, one of six cytochrome P450 genes previously examined in camel tissues by western blotting and semi-quantitative PCR. The Camelus dromedarius CYP2E1 has an open reading frame of 1,473 bp, and the cDNA encodes a protein of 490 amino acid residues with a molecular weight of 54.8 kDa. The deduced amino acid sequence showed the highest identity with Bos taurus (88%), Sus scrofa (87%), and Homo sapiens (83%). In a phylogenetic analysis, the C. dromedarius CYP2E1 isoform was located beside cattle and pigs. The deduced amino acid sequence of camel CYP2E1 showed the conserved proline-rich amino terminus and the heme-binding signature localized near the carboxy terminus of the protein.     

Genetic differences between Tunisian camel and sheep strains of the cestode Echinococcus granulosus revealed by SSCP

Ovine and dromedary Echinococcus granulosus isolates from Tunisia were identified as G1 and G6 strains based on polymorphism of the mitochondrial cytochrome C oxydase CO1. Single strand conformation polymorphism (SSCP) was used in order to examine the genetic variation within and between Tunisian G1 and G6 strains and to estimate the extent of selfing. The dromedary isolates are genetically distinct from sheep isolates (high value of genetic variation between populations: Fst= 0.46). No significant deficiency in heterozygotes was found in sheep isolates, whereas heterozygote deficiency (suggesting selfing) was found in a limited number of camel isolates.     

Flavin-containing monooxygenase activity in camel tissues: comparison with rat and human liver enzymes

We previously reported the occurrence of multiple forms of drug metabolizing enzymes in camel tissues. In this study, we demonstrated for the first time, flavin-containing monooxygenase (FMO)-dependent metabolism of two model substrates methimazole (MEM) and N,N'-dimethylaniline (DMA) by camel liver, kidney, brain and intestine. FMO-catalyzed metabolism in the microsomes of camel tissues was independent of cytochrome P450 (CYP) activity and exhibited a pH and temperature dependence characteristic of FMO enzymes. Use of inhibitors of CYP activities, SKF525A, octylamine or antibody against NADPH-P450 reductase, did not significantly alter the FMO-dependent substrate metabolism. Using MEM as a model substrate for FMO activity, we show that camel liver has an activity similar to that in rat and human livers. MEM metabolism in extrahepatic tissues in camels was significantly lower (60%-80%) than that in liver. Our results suggest occurrence of FMO in camel tissues, with catalytic properties similar to those in rat and human livers. These results may help in better understanding the effects of pharmacologically and toxicologically active compounds administered to camels.     

Disaccharidase activities in camel small intestine: biochemical investigations of maltase-glucoamylase activity

Disaccharidases (maltase, cellobiase, lactase, and sucrase), alpha-amylase, and glucoamylase in the camel small intestine were investigated to integrate the enzymatic digestion profile in camel. High activities were detected for maltase and glucoamylase, followed by moderate levels of sucrase and alpha-amylase. Very low activity levels were detected for lactase and cellobiase. Camel intestinal maltase-glucoamylase (MG) was purified by DEAE-Sepharose and Sephacryl S-200 columns. The molecular weight of camel small intestinal MG4 and MG6 were estimated to be 140,000 and 180,000 using Sephacryl S-200. These values were confirmed by SDS-PAGE, where the two enzymes migrated as single subunits. This study encompassed characterization of MGs from camel intestine. The Km values of MG4 and MG6 were estimated to be 13.3 mM and 20 mM maltose, respectively. Substrate specificity for MG4 and MG6 indicated that the two enzymes are maltase-glucoamylases because they catalysed the hydrolysis of maltose and starch with alpha-1,4 and alpha-1,6 glycosidic bonds, but not sucrose with alpha-1,2 glycosidic bond which was hydrolyzed by sucrase-isomaltase. Camel intestinal MG4 and MG6 had the same optimum pH at 7.0 and temperature optimum at 50 degrees C and 40 degrees C, respectively. The two enzymes were stable up to 50 degrees C and 40 degrees C, followed by strong decrease in activity at 60 degrees C and 50 degrees C, respectively. The effect of divalent cations on the activity of camel intestinal MG4 and MG6 was studied. All the examined divalent cations Ca(2+), Mn(2+), Mg(2+), Co(2+) and Fe(3+) had slight effects on the two enzymes except Hg(2+) which had a strong inhibitory effect. The effect of different inhibitors on MG4 and MG6 indicated that the two enzymes had a cysteine residue.     

Variability of Some Milk-Associated Genes and Proteins in Several Breeds of Saudi Arabian Camels

To gain knowledge on the molecular basis of diversity of several clans of Saudi camel (Camelus dromedarius) characterization of these animals was conducted at both genetic and protein levels. To this end, blood and milk samples were collected from several camel breeds at different Saudi Arabia locations (northern Jeddah, Riyadh, and Alwagh governorates). Genomic DNA was extracted from blood of four Saudi camel breeds (Majahem, Safra, Wadha, and Hamara), and DNA fragments of the casein and α-lactalbumin genes were amplified. The retrieved DNA sequences were analyzed for genetic variability. The inter-simple sequence repeat technique was used for confirming the relationships among the analyzed camel breeds, and the PCR–RFLP with two restriction enzymes was utilized for exploring their molecular variations. The number of haplotypes, gene diversity, nucleotide diversity, average number of nucleotide differences, and sequence conservation were calculated for all the analyzed DNA sequences. These analyses revealed the presence of several single nucleotide polymorphisms in the analyzed DNA sequences. A group of neighbor joining trees was built for inferring the evolutionary variations among the studied animals. Protein profiling of milk from different camel clans was also conducted, and differences between and within the Saudi camel clans were easily found based on the isoelectric focusing (IEF) profiles using ampholytes with different IEF range. This study revealed that analyzed camel breeds show low levels of genetic differences. This may be a reflection of the evolutionary history of C. dromedarius that was domesticated based on a highly homogeneous ancestor ecotype.     

Studies on camel hemoglobin. 1. Physico-chemical properties and some structural aspects of camel hemoglobin (Camelus dromedarius).

Hemoglobin from an adult camel (Camelus dromedarius) was prepared from the red cell lysate by CM- and DEAE-cellulose chromatography. The purified hemoglobin showed a lesser mobility on starch gel electrophoresis at pH 8.5 than that of human hemoglobin C. Native camel hemoglobin contains 95-99% alkali-resistant hemoglobin and in soluble in 2.94 M K2HPO4/KH2PO4 buffer. Different forms of camel hemoglobin show similar ammonium sulfate precipitation curves. Indirect evidence for the stability of camel hemoglobin solutions was obtained from several sources. Spontaneous met-hemoglobin formation is extremely slow and minimal quantities of degradation products appear on starch gel electrophoresis and on chromatographic separation. The alpha and beta chains of camel hemoglobin A were separated on a CM-23 column by the use of a pyridine formate gradient. Large peptide fragments were obtained by tryptic digestion of maleylated alpha and beta chains. The N-terminal structure of the alpha and beta chains and of tryptic maleylated peptides derived from alpha and beta chains are presented. Between adult camel hemoglobin and adult human hemoglobin six amino acid differences in the N-terminal 20 amino acid residues of the alpha chain, at residues: 4, 5, 12, 14, 17, and 19; eight amino acid substitutions were found in the beta chain at positions: 4, 5, 6, 9, 12, 13, 16, and 19. Substitutions at alpha5 Ala leads to Lys, and beta19 Asn leads to Lys, increase the net positive charge of camel hemoglobin by two, while other substitutions result in no charge differences. The molecular basis of the stability of camel adult hemoglobin is discussed.     

Semen cryopreservation in Bactrian camel (Camelus bactrianus) using SHOTOR diluent: effects of cooling rates and glycerol concentrations

Experiments were conducted with a final goal of providing a suitable protocol for cryopreservation of Bactrian camel semen. In Experiment I, the effect of average cooling rate (slow cooling: 0.14 versus fast cooling: 0.55 degrees C/min) on the viability of chilled semen was evaluated. In Experiment II, the effect of different concentrations of glycerol (4, 6 and 8%) on the post-thaw viability of frozen sperm was investigated. In Experiment III, the efficiency of SHOTOR diluent was compared with IMV buffers for the cryopreservation of camel semen. Viability parameters including progressive forward motility (PFM), plasma membrane integrity and percentage of live spermatozoa were assessed. Progressive forward motility of sperm cooled at the faster rate was superior after incubating for 24h at 4 degrees C compared to that cooled at the slower rate (P<0.05). Post-thaw viability of Bactrian camel sperm was better using a final glycerol concentration of 6% compared to 4 and 8% (P<0.05). Progressive forward motility of frozen-thawed sperm was greater using SHOTOR diluent (29.9%) compared to IMV buffers (4.2%, P<0.05). In conclusion, semen cryopreservation in Bactrian camel is feasible when it is extended in SHOTOR diluent, cooled within 1h (average cooling rate: 0.55 degrees C/min) to 4 degrees C, and then exposed to glycerol, at the final concentration of 6%.     

A comparative study of ketone body metabolism between the camel (Camelus dromedarius) and the sheep (Ovis aries).

1. Plasma levels of beta-hydroxybutyrate (BHB), and acetoacetate (AcAc) have been measured in camels (Camelus dromedarius) and sheep (Ovis aries). The activity of beta-hydroxybutyrate dehydrogenase (BHB-deH2) (E.C. 1.1.1.30) was studied in the rumen epithelium and the liver of these animals. 2. Concentrations of plasma BHB and AcAc in the camel were in respective order 33 and 4 times lower than that of the sheep. The ratios of BHB to AcAc were 0.61 and 4.8 for the camel and sheep, respectively. 3. The activity of BHB-deH2 in the rumen epithelium of the camel and sheep were 7.15 and 66 mumol/hr/g wet wt tissue, respectively. The activity in both species was higher in the rumen epithelium than in the liver.     

Carbohydrases in camel (Camelus dromedarius) pancreas. Purification and characterization of glucoamylase

The present study analyzed the existence of carbohydrases in camel pancreas compared to some other ruminants. Disaccharidases (maltase, cellobiase, lactase, trehalase and sucrase), glucoamylase and alpha-amylase were detected in pancreas of camel, sheep, cow and buffalo. Enzyme levels in sheep were lower than in the other ruminants. The highest level was detected for alpha-amylase (EC 3.2.1.2). Moderate activity levels were detected for glucoamylase (EC 3.2.1.3) and maltase (EC 3.2.1.20), while other disaccharidases showed very low activity. The results suggested that, in addition to alpha-amylase, glucoamylase and maltase may be synthesized and secreted from pancreas to the small intestine in ruminants. Camel pancreatic glucoamylase was purified and characterized. The purification procedure included glycogen precipitation and chromatography on DEAE-Sepharose and Sepharose 6B. The molecular mass was 58 kDa for native and denatured enzyme using gel filtration and SDS-PAGE, respectively. The enzyme had a pH optimum at 5.5 and a Km of 10 mg starch/mL with more affinity toward potato soluble starch than the other carbohydrates. Glucoamylase had a temperature optimum at 50 degrees C with heat stability up to 30 degrees C. The effect of different cations and inhibitors was examined. The camel pancreatic glucoamylase may possess an essential thiol.     

Amino acids content and electrophoretic profile of camel milk casein from different camel breeds in Saudi Arabia

This study aimed to evaluate amino acids content and the electrophoretic profile of camel milk casein from different camel breeds. Milk from three different camel breeds (Majaheim, Wadah and Safrah) as well as cow milk were used in this study.Results showed that ash and moisture contents were significantly higher in camel milk casein of all breeds compared to that of cow milk. On the other hand, casein protein of cow milk was significantly higher compared to that of all camel milk breeds. Molecular weights of casein patterns of camel milk breeds were higher compared to that of cow milk.Essential (Phe, Lys and His) and non-essential amino acids content was significantly higher in cow milk casein compared to the casein of all camel milk breeds. However, there was no significant difference for the other essential amino acids between cow casein and the casein of Safrah breed and their quantities in cow and Safrah casein were significantly higher compared to the other two breeds. Non-essential amino acids except Arg and the essential amino acids (Met, Ile, Lue and Phe) were also significantly higher in cow milk α-casein compared to α-casein from all camel breeds. Moreover, essential amino acids (Val, Phe and His) and the non-essential amino acids (Gly and Ser) content was significantly higher in cow milk β-casein compared to the β-casein of all camel milk breeds and the opposite was true for Lys, Thr, Met and Ile. However, Met, Ile, Phe and His were significantly higher for β-casein of Majaheim compared to the other two milk breeds. The non-essential amino acids (Gly, Tyr, Ala and Asp) and the essential amino acids (Thr, Val and Ile) were significantly higher in cow milk κ-casein compared to that for all camel milk breeds. There was no significant difference among all camel milk breeds in their κ-casein content of most essential amino acids.Relative migration of casein bands of camel milk casein was not identical. The relative migration of α_s-, β- and κ-casein of camel casein was slower than those of cow casein. The molecular weights of α_s-, β- and κ-casein of camel caseins were 27.6, 23.8 and 22.4 KDa, respectively. More studies are needed to elucidate the structure of camel milk.     

Extraction and characterization of gelatin from camel skin (potential halal gelatin) and production of gelatin nanoparticles

Gelatin is used as an ingredient in both food and non-food industries as a gelling agent, stabilizer, thickener, emulsifier, and film former. Porcine skins, bovine hides, and cattle bones are the most common sources of gelatin. However, mammalian gelatins are rejected by some consumers due to social, cultural, religious, or health-related concerns. In the present study, gelatin was obtained from camel skin as an alternative source using a combination of processing steps. Central composite design combined with response surface methodology was used to achieve high gelatin yields under different extraction conditions: temperatures of 40, 60, and 80 °C; pH values of 1, 4, and 7; and extraction times of 0.5, 2.0, and 3.5 min. Maximum gelatin yield from camel skin (29.1%) was achieved at 71.87 °C and pH 5.26 after 2.58 min. The extracted gelatin samples were characterized for amino acid profile, foaming capacity, film formation, foam stability, and gel strength (Bloom value). Gelatin nanoparticles were produced, and their morphology and zeta potential were determined. Bloom value of the camel skin gelatin was 340 g. Amino acid analysis revealed that the extracted gelatin showed high glycine and proline contents. Analysis of camel skin gelatin nanoparticle and functional properties revealed high suitability for food and non-food applications, with potential use in the growing global halal food market.     

Genotypic characterization of Iranian camel (Camelus dromedarius) isolates of Echinoccocus granulosus

In the present study, Echinoccocus granulosus isolates collected from camels (Camelus dromedarius) in eastern Iran were characterized based on the nucleotide sequences of mitochondrial CO1 and NDI genes. The molecular results for camel isolates demonstrated that at least 2 different genotypes are present, i.e., a buffalo genotype (G3) and the camel genotype (G6). Although the sequences of the Iranian camel genotype (G6) are completely homologous to the reference sequence of G6 (M84666) of E. granulosus , a nucleotide mutation (C to T at position 168) was detected in the CO1 sequences of the Iranian G3 isolates (HM626405) when compared with the reference G3 genotype (M84663). The findings of the present study represent the demonstration of the buffalo strain in camels. As previously reported, humans can be infected by this genotype; accordingly, the epidemiological importance of this genotype in the camel population should be considered in further studies.     

The distribution of phosphofructokinase isoenzymes in the liver of camel, rat and rabbit

1. The distribution of phosphofructokinase isoenzymes have been compared among camel, rat and rabbit livers. 2. Only a single phosphofructokinase isoenzyme is present in the camel liver which has shown different physical and regulatory properties from the isoenzymes of rat and rabbit liver. 3. The ammonium sulphate precipitation curves of the camel and rabbit enzymes were monophasic, whereas the rat enzyme was biphasic. 4. Rabbit liver phosphofructokinase was slightly more anodic than the rat enzyme, whereas the camel enzyme was the least anodic as shown by the techniques of DEAE-cellulose chromatography and cellulose acetate electrophoresis. 5. Partially purified camel liver phosphofructokinase showed different regulatory properties from the rabbit and rat isoenzymes as the apparent Km values were 0.58, 0.45 and 0.82 mM respectively.     

Immunochemical characteristics of the ovine polymeric haptoglobin HpC

It was found that haptoglobins of camel, cattle, horse, pig, rat, guinea pig and man form upon immunoelectrophoresis precipitation arcs with antibodies against polymeric sheep haptoglobin C, corresponding to alpha 2-globulins. The immunocrossreactivity of haptoglobins of man and various animal species towards antibody to sheep haptoglobin (100, 88.0, 75.2, 72.1, 56.3, 51.0, 41.3 and 28.0% for haptoglobin of sheep, camel, pig, cattle, man, rat, guinea pig and horse, respectively) was determined. The intensity of crossreactions between sheep haptoglobin and the proteins under study towards antibody to haptoglobin C reflects the similarity of their primary structure and, consequently, the immune homology of their molecules. Using quantitative titration, the antigenic valency values for human (6), sheep (5), cattle (4) and horse (3) haptoglobins were determined.     

Purification and partial characterization of camel (Camelus Dromedarius) ceruloplasmin

Adult and young camel ceruloplasmin (Cp) were isolated and purified using the single-step chromatography on amino ethyl-activated sepharose. There are no differences between the adult and the young camel protein. The molecular mass of the protein, as estimated by SDS-PAGE (denaturant conditions), was approximately 130000 Da. The electrophoretic mobility of camel Cp is slightly higher as compared to human and sheep protein suggesting that the camel Cp is homogeneous, compact and more acid. The copper content was estimated to be 5.8+/-0.3 atoms per molecule. The spectroscopic feature includes an absorption maximum at 610 nm, which could be attributed to type 1 copper. The EPR spectrum was completely devoid of any typical signal of the type 2 copper. The kinetic parameters of the adult camel Cp for the specific activity as p-phenylendiamine oxidase were determined as K(m)=0.42 mM and V(max)=0.93 microM NADH/mn/mg Cp. The optimum pH for the activity was 5.7.     

Effectiveness of a tris-based extender (SHOTOR diluent) for the preservation of Bactrian camel (Camelus bactrianus) semen

The development of a suitable semen extender is required to extend artificial breeding programs and to preserve the genetic potential of Bactrian camel. Experiments were conducted to provide the optimal osmolality and pH of tris-based extender and to compare that with available extenders for short-term preservation of Bactrian camel semen at 4 degrees C during 24 h. In experiments I and II, the effects of varying osmolalities (270, 300, 330, 360, and 390 mOsm/kg) and pHs (5.5, 6, 6.9, 7.5, 7.9, and 8.9) of tris-based extender on sperm viability were investigated. In experiment III, the efficiency of tris-based extender (SHOTOR diluent) in preserving Bactrian camel semen was compared with lactose (10%), sucrose (10%) and Green buffer. Viability parameters including progressive forward motility (PFM), plasma membrane integrity and the percentage of live spermatozoa were assessed. The data were analyzed using general linear model procedure. In the majority of assessments using tris-based extender, the viability of spermatozoa was superior at the osmolality of 330 mOsm/kg and pH of 6.9. PFM was significantly greater at the time of semen dilution in tris-based (65.5%) and Green buffer (60.5%) compared to that of lactose (31%) and sucrose (28%) extenders (P<0.05), and remained elevated throughout the experiment. There was no significant difference in other viability parameters among 4 extenders (P>0.05). In conclusion, the utilization of a tris-based extender, having the osmolality of 330 mOsm/kg and pH of 6.9, favors the short-term preservation of the Bactrian camel spermatozoa under chilled condition.     

NADPH-dependent reduction of glyceraldehyde: a unusually high activity in the lens of the camel (Camelus dromedarius)

The enzymes of the polyol pathway, namely aldose reductase and sorbitol dehydrogenase, were measured in camel lens extracts. A NADPH-dependent glyceraldehyde and erythrose reductase activity 25 times higher than that of calf lens was observed in camel lens. A preliminary comparison between this enzyme activity present in the camel and aldose reductase of calf lens is reported.