Redox homeostasis and respiratory metabolism in camels (Camelus dromedaries): comparisons with domestic goats and laboratory rats and mice

We have previously reported the occurrence of multiple forms of drug-metabolizing enzymes in camel tissues. Here, we investigate glutathione (GSH)-dependent redox homeostasis, reactive oxygen species (ROS) production and mitochondrial respiratory functions in camel tissues and compare them with imported domestic goats and laboratory rats and mice. Cytochrome P450 2E1 (CYP 2E1) and GSH-metabolizing enzymes were differentially expressed in the liver and kidney of these animals. Camel liver has significantly lower GSH pool than that in goats, rats and mice. Mitochondria isolated from the tissues of these animals showed a comparable ability to metabolize specific substrates for respiratory enzyme complexes I, II/III and IV. These complexes were metabolically more active in the kidney than in the liver of all the species. Furthermore, the activity of complex IV in camel tissues was significantly lower than in other species. On the other hand, complex II/III activity in camel kidney was higher compared to the other species. In addition, as expected, we observed that inhibitors of these enzyme complexes augment the production of mitochondrial ROS in camel and goat tissues. These results help to better understand the metabolic ability and adaptation in desert camels in comparison with domestic goats and laboratory rats and mice since they are exposed to different environmental and dietary conditions. Our study may also have implications in the pharmacology and toxicology of drugs and pollutants in these species.     

The house musk shrew (Suncus murinus): a unique animal with extremely low level of expression of mRNAs for CYP3A and flavin-containing monooxygenase

Expression of drug-metabolizing enzymes including cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) in various tissues of Suncus murinus (Suncus) were examined. Northern blot analysis showed that mRNAs hybridizable with cDNAs for rat CYP1A2, human CYP2A6, rat CYP2B1, human CYP2C8, human CYP2D6, rat CYP2E1, human CYP3A4 and rat CYP4A1 were expressed in various tissues from Suncus. The mRNA level of CYP2A in the Suncus lung was very high. Furthermore, it was found that the level of CYP2A mRNA in the Suncus lung was higher compared to the Suncus liver. The expression level of mRNA hybridizable with cDNA for human CYP3A4 was very low. The presence of CYP3A gene in Suncus was proven by the induction of the CYP with dexamethasone. Very low expression levels of mRNAs hybridizable with cDNAs for rat FMO1, rat FMO2, rat FMO3 and rat FMO5 were also seen in Suncus liver. No apparent hybridization band appeared when human FMO4 cDNA was used as a probe. The hepatic expression of mRNAs hybridizable with cDNAs for UDP-glucuronosyltransferase 1*6, aryl sulfotransferase, glutathione S-transferase 1, carboxyesterase and microsomal epoxide hydrolase in the Suncus were observed. These results indicate that the Suncus is a unique animal species in that mRNAs for CYP3A and FMO are expressed at very low levels.     

Tissue Specific Expression of Glutathione S-transferases, Glutathione Content and Lipid Peroxidation in Camel Tissues

Differential expression of glutathione S-transferase (GST) enzyme activity in various tissues of the camel was observed with a maximum activity in the liver. Compared with the rat and human livers, GST activity in camel liver was 50% lower than that of rat liver and similar to that of human liver. Extrahepatic tissues in camel have a comparable GST activity with those of similar tissues in the rat. Assay of GST activity using ethacrynic acid as substrate demonstrated maximum activity in the camel brain followed by intestine, liver and kidney. Microsomal GST activity in camel tissues was expressed in the order of liver > testis > intestine ≈ kidney ≈ brain. Phenotyping of GST was performed in camel hepatic and extrahepatic tissues using human specific antibodies to class α, μ, and π cytosolic GST isoenzymes and rat specific antibody to the microsomal GST. Western immunoblot and immunohistochemical analyses showed an abundant expression of GST α and μ in the camel liver, while π was very poorly expressed. Camel extrahepatic tissues however, had a significant expression of GST π. The camel GST isoenzymes were found to be predominantly expressed in the hepatocytes around the central vein with a gradual decrease in expression in the hepatocytes located toward the periphery. Kidney cortex exhibited a greater expression of the enzyme protein in the proximal tubules as compared to the glomeruli. Glutathione (GSH) concentration in rat tissues, except in the brain, was about 2-fold higher than that of camel tissues. Rate of NADPH-dependent microsomal lipid peroxidation was comparable both in the rat and camel tissues with the highest activity in the brain and lowest activity in the intestine. The differential expression of GST isoenzymes in different organs of the camel, GSH concentration and the rate of lipid peroxidation in different tissues may be important factors in determining the differential susceptibility of camel tissues to the toxic effects of xenobiotics.     

Characterization of Phase I and Phase II hepatic drug metabolism activities in a panel of human liver preparations.

The role of drug metabolism in drug discovery (lead compound selection) and the traditional role of identifying the enzymes involved in biotransformation pathways (reaction phenotyping) have both relied heavily on the availability and use of a human liver bank. The assessment of drug metabolizing enzyme activity and variability in a series of individual human livers is essential when characterizing the enzymes involved in metabolic pathways (i.e. correlation analysis). In this regard, a human liver bank of 21 samples (14 males, six females, and one unknown) was characterized with respect to the activity of several important drug metabolizing enzymes. The total CYP450 content of the livers ranged from 0.06 to 0.46 nmol/mg microsomal protein. The fold variations found in specific enzyme contents were as follows: CYP1A2 (3x), CYP2A6 (21x), CYP2C9 (8x), CYP2C19 (175x), CYP2D6 (18x), CYP2E1 (5x), CYP3A4 (18x), FMO (2.5x), UDPGT (4x), NAT (7x), COMT (5x), ST (5x), TPMT (3x), and GST (2.5x). In general, the fold variation of the Phase II enzymes was lower compared with the Phase I enzymes, with the exceptions of CYP1A2, CYP2E1, and FMO. Similar data were reviewed from other established liver banks and compared with regard to the relative variability observed in drug metabolizing capacities found in this study.     

Diversity and distribution of CYP gene family in Bactrian camel

Cytochrome P450 (CYP) enzymes belong to a superfamily of monooxygenases which are phase I enzymes responsible for the first pass metabolism of about 90% of drugs in animals. However, these enzymes are often polymorphic and metabolism of the same drug in different species or different individuals is influenced by genetic and non-genetic factors. Bactrian camels are capable of survival in harsh living environments, being able to consume diets that are often toxic to other mammals and can tolerate extreme water and food deprivation. The aim of this study was to investigate whether the Bactrian camel’s special metabolic pathways and unique detoxification capabilities are attributable to particularities of the CYP gene family. The Bactrian camel’s whole genome sequencing data were systemically analyzed and annotated, and then, CYP gene family was searched from the whole protein database and compared with CYP gene families of cattle, horse, chicken, and human. The total of 63 CYP gene copies were found in Bactrian camel’s whole genome and were classified into 17 families and 38 subfamilies. Among them, 9 multi-gene families were found, and CYP2, CYP3, and CPY4 have 27, 6, and 7 subfamilies, accounting for 43, 10, and 11% in camel CYP gene, respectively. In comparison with cattle, chicken, horse, and human, the distribution of CYP gene subfamilies in camel is different, with more CYP2J and CYP3A copies in the Bactrian camel, which may contribute to the Bactrian camel’s specific biological characteristics and metabolic pathways. Comparing to the cow, horse, chicken, and human CYP genes, the distribution of CYP gene subfamilies is distinct in the Bactrian camel. The higher copy number of CYP2J gene and CYP3A gene in Bactrian camel may be the important factors contributing to the distinct biological characteristics and metabolic pathways of Bactrian camels for adaptation to the harsh environments.     

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.     

Immunochemical characterization and toxicological significance of P-450HFLb purified from human fetal livers.

Immunochemical properties of P-450HFLb purified from human fetal livers were investigated. P-450HFLb cross-reacted with antibodies to rat P-4501A1 but not with antibodies to CYP2A6, CYP2C9, CYP3A7 (P-450HFLa) and rat CYP2B1. In addition, P-450HFLb also cross-reacted with both monospecific antibodies to rat CYP1A1 and CYP1A2. However, P-450HFLb was shown to be an immunochemically distinct form of cytochrome P-450 from P-450PA (human CYP1A2). Immunoblot analysis of human fetal livers with the antibodies to P-450HFLb showed that P-450HFLb was expressed in all fetal livers studied although there appeared to be individual differences in the amounts of P-450HFLb expressed in fetal livers. The formation of mutagens from IQ (but not from AFB1) in fetal liver homogenates was inhibited by the antibodies to P-450HFLb in a dose dependent manner. These results suggest that P-450HFLb may be a form of human cytochrome P-450 classified into CYP1 gene family, and that the cytochrome P-450 is, in part, responsible for the mutagenic activation of IQ in human fetal livers as well as CYP3A7 (P-450HFLa).     

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.     

The CYP2A3 gene product catalyzes coumarin 7-hydroxylation in human liver microsomes

Three cDNAs, designated IIA3, IIA3v, and IIA4, coding for P450s in the CYP2A gene subfamily were isolated from a lambda gt11 library prepared from human hepatic mRNA. Only three nucleotide differences and a single amino acid difference, Leu160----His, were found between IIA3 and IIA3v, indicating that they are probably allelic variants. IIA4 displayed 94% amino acid similarity with IIA3 and IIA3v. The three cDNAs were inserted into vaccinia virus, and recombinant viruses were used to infect human hepatoma Hep G2 cells. Only IIA3 was able to produce an enzyme that had a reduced CO-bound spectrum with a lambda max at 450 nm. This expressed enzyme was able to carry out coumarin 7-hydroxylation (turnover number of 15 min-1) and ethoxycoumarin O-deethylation. cDNA-expressed IIA3v and IIA4 failed to incorporate heme and were enzymatically inactive. Analysis of IIA proteins in human liver microsomes, using antibody against rat IIA2, revealed two proteins of 49 and 50 kDa, the former of which appeared to correlate with human microsomal coumarin 7-hydroxylase activity. A more striking correlation was found between IIA mRNA and enzyme activity. The rat antibody was able to completely abolish coumarin 7-hydroxylase activity in 12 liver samples. In addition, kinetics of coumarin metabolism in two livers were monophasic over the substrate concentration tested. Km values obtained from human liver (2.3 microM) were similar to those obtained from lysates of hepatoma cells expressing IIA3 (3.6-7.1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and tissue distribution of a novel human cytochrome p450 of the CYP3A subfamily, CYP3A43

On the basis of the detection of an expressed sequence tag ('EST') similar to the human cytochrome P450 3A4 cDNA, we have identified a novel member of the human cytochrome P450 3A subfamily. The coding region is 1512-bp long and shares 84, 83, and 82% sequence identity on the cDNA level with CYP3A4, 3A5, and 3A7, respectively, with a corresponding amino acid identity of 76, 76, and 71%. Quantitative real time based mRNA analysis revealed CYP3A43 expression levels at about 0.1% of CYP3A4 and 2% of CYP3A5 in the liver, with significant expression in 70% of the livers examined. Gene specific PCR of cDNA from extrahepatic tissues showed, with the exception of the testis, only low levels of CYP3A43 expression. The CYP3A43 cDNA was heterologously expressed in yeast, COS-1 cells, mouse hepatic H2.35 cells and in human embryonic kidney (HEK) 293 cells, but in contrast to CYP3A4 which was formed in all cell types, no detectable CYP3A43 protein was produced. This indicates a nonfunctional protein or specific conditions required for proper folding. It is concluded that CYP3A43 mRNA is expressed mainly in liver and testis and that the protein would not contribute significantly to human drug metabolism.     

A comparison of aroclor 1254-induced and uninduced rat liver microsomes to human liver microsomes in phenytoin O-deethylation, coumarin 7-hydroxylation, tolbutamide 4-hydroxylation, S-mephenytoin 4'-hydroxylation, chloroxazone 6-hydroxylation and testosterone 6beta-hydroxylation

Aroclor 1254-induced rat liver homogenate supernatant (liver S-9) is routinely used as an exogenous metabolic activation system for the evaluation of mutagenicity of xenobiotics. The purpose of this study is to evaluate whether results obtained with Aroclor 1254-induced liver microsomes would be relevant to human. Aroclor 1254-induced and uninduced rat liver microsomes were compared to human liver microsomes in the metabolism of substrates which are known to be selectively metabolized by the major human cytochrome P450 (CYP) isoforms. The activities studied and the major CYP isoforms involved were as follows: phenacetin O-deethylation (CYP1A2); coumarin 7-hydroxylation, (CYP2A6); tolbutamide 4-hydroxylation (CYP2C9), S-mephenytoin 4'-hydroxylation (CYP2C19); dextromethorphan O-demethylation (CYP2D6); chloroxazone 6-hydroxylation (CYP2E1); and testosterone 6beta-hydroxylation (CYP3A4). We found that both induced and uninduced rat liver microsomes were active in all the pathways studied with the exception of coumarin 7-hydroxylation. Coumarin 7-hydroxylation was observed with human liver microsomes but not the rat liver microsomes. Aroclor-1254 was found to induce all activities measured, with the exception of coumarin 7-hydroxylation. Dextromethorphan O-deethylation activity was higher in the rat liver microsomes than the human liver microsomes. Testosterone 6beta-hydroxylation activity was found to be similar between the human liver microsomes and the induced rat liver microsomes. Our results suggest that experimental data obtained with Aroclor 1254-induced rat liver microsomes may not always be relevant to human.     

Antioxidant levels in tissues of young and adult camels (Camelus dromedarius)

In this study, we measured the concentration of some antioxidant substances in erythrocytes hemolysate, liver, kidney and brain in young and adult camels. It has been found that the activity of the antioxidant enzymes glutathione peroxidase (GSH-Px), catalase (CAT), superoxide dismutase (SOD) and the concentration of glutathione, ascorbic acid and alpha-tocopherol are high in both young and adult camels. GSH-Px and CAT activities were higher in adult camels than in the young whereas no significant difference in the activity of SOD between young and adult camels was noticed. Glutathione was present in all tissues studied. Ascorbic acid was found to have significantly higher values in young camels. From this study it could be concluded that, as in other mammals, camel tissues contain a powerful antioxidant system. The liver has the highest contents of antioxidants and antioxidant enzymes indicating that it plays an important role in pro-oxidants detoxification. Age has a variable effect on the antioxidant system in camels.     

Some distinctions between flavin-containing and cytochrome P450 monooxygenases

This minireview summarizes information concerning the differences and similarities of the human flavin-containing- (FMO, E.C. 1.14.13.8) and the cytochrome P450-monooxygenases (CYP, E.C. 1.14.14.1). Human FMO oxygenates soft nucleophiles. CYP mainly catalyzes C-H abstraction but also oxidizes nitrogen- and sulfur-containing compounds. Both FMO and CYP generally convert lipophilic compounds into more hydrophilic metabolites. The mechanism by which each monooxygenase operates is quite distinct. Sometimes, CYP or FMO bioactivate chemicals to reactive metabolites but to date, drug toxicity thus far observed in the clinic is mainly the result of CYP-dependent oxidation. Both FMO and CYP possess genetic variability that may contribute to inter-individual variability observed for drug metabolism. In contrast to CYP, FMO is not induced or readily inhibited and potential adverse drug-drug interactions are minimized for drugs prominently metabolized by FMO. These properties may provide advantages in drug design, and by incorporating FMO detoxication pathways into drug candidates, more drug-like materials may emerge.     

Impact of warm ischemic time on microsomal P450 isoforms in a porcine model of therapeutic liver resection

Human microsomes and hepatocytes obtained from non-transplantable livers of brain-dead donors are very useful in predicting the in vivo metabolism of xenobiotics in humans. Fresh liver specimens obtained from therapeutic liver resection are also useful for research in cases where non-transplantable livers are not readily available. In the present study, the effect of warm ischemic duration, in the course of hepatic surgery, on the activities of liver cytochrome P450 (CYP) CYP1A, CYP2C, CYP2D, CYP2E1 and CYP3A were evaluated in a porcine model. Partial occlusion (portal vein and hepatic artery occlusion) decreased the activities of CYP2C, CYP2E and CYP3A, but not those of CYP1A and CYP2D. CYP3A, known to account for an average 30% of total P450 content in the human liver was the most susceptible to the warm ischemia. These results demonstrate that the activities of CYP isoforms, particularly those of CYP3A, are markedly affected by warm ischemia; it is, therefore, essential that care should be exercised when using microsomes prepared from surgically removed livers.     

Evidence for cytochrome P450 3A expression and catalytic activity in rat blood lymphocytes

Freshly isolated peripheral blood lymphocytes from control rats were found to catalyze the N-demethylation of erythromycin, known to be mediated by cytochrome P450 3A (CYP3A) isoenzymes in rat liver. Pretreatment of rats with dexamethasone (100 mg/kgx3 days, i.p.), a CYP3A inducer, resulted in 3-4-fold increase in the activity of erythromycin demethylase (EMD) in freshly isolated peripheral blood lymphocytes. This increase in the enzyme activity was found to be associated with an increase in the rate of the reaction and affinity of the substrate towards the enzyme. Significant inhibition of the EMD activity on in vitro addition of ketoconazole, a specific CYP3A inhibitor in liver and polyclonal antibody raised against rat liver CYP3A have suggested that EMD activity in blood lymphocytes is catalyzed primarily by CYP3A isoenzymes. Further, immunoblot analysis with polyclonal antibody raised against rat liver CYP3A revealed significant immunoreactivity, co-migrating with the liver isoenzyme, indicating constitutive expression of CYP3A in blood lymphocytes. Pretreatment with dexamethasone was found to significantly increase the expression of CYP3A protein in freshly isolated rat blood lymphocytes, as observed with liver. Likewise, significant CYP3A mRNA detected in control rat blood lymphocytes has further demonstrated constitutive expression of CYP3A isoenzymes in blood lymphocytes. Furthermore, several fold increase in CYP3A mRNA expression following pretreatment with dexamethasone showed similarities in the regulation of CYP3A isoenzymes in rat blood lymphocytes with the liver enzyme. The data suggest that the blood lymphocytes can be used to monitor tissue expression of CYP3A isoenzymes and validate the suitability of lymphocytes as surrogates of CYP status in less accessible target tissues.     

Interindividual differences in hepatic expression of CYP3A4: relationship to genetic polymorphism in the 5'-upstream regulatory region.

Cytochrome P450 3A4, the most abundant P450 form in human liver, exhibits a very broad substrate specificity and is of great importance for drug metabolism. The interindividual difference in the hepatic expression of CYP3A4 is considerable. In order to investigate possible genetic factor(s) causing this variation, the rate of 6beta-hydroxylation of testosterone in human liver microsomes prepared from 46 different human liver samples was determined and the 5'upstream region (+10 to -490 bp) was sequenced from genomic DNA isolated from 39 of these livers. We found a 31-fold variation of the testosterone hydroxylase activity between the samples. However, a very high sequence homology between the CYP3A4 5'-upstream regions sequenced from the 78 different alleles was found. In fact, only three variant nucleotide exchanges were identified, all causing a -290 A-->G mutation (CYP3A4-V) in a so called nifedipine specific element (NFSE). The importance of this element and the polymorphism was evaluated by gel shift analysis. Competition experiments revealed that the binding of nuclear proteins, although having lower affinity to the CYP3A4-V form of the element, was unspecific in nature. In accordance, no influence of this polymorphism was seen on the microsomal testosterone hydroxylase activity in vitro. It is concluded that the promoter region of CYP3A4 is highly conserved, the only polymorphism being in the NFSE, which however does not influence the enzyme expression in liver to a significant degree. This casts doubt of a previously described relationship between the CYP3A4-V allele and cancer in the prostate and leukaemia.     

An inhibition study of beauvericin on human and rat cytochrome P450 enzymes and its pharmacokinetics in rats

Beauvericin is a secondary metabolite natural product from microorganisms and has been shown to have a new potential antifungal activity. In this study, the metabolism and inhibition of beauvericin in human liver microsomes (HLM) and rat liver microsomes (RLM) were investigated. The apparent K(m) and V(max) of beauvericin in HLM were determined by substrate depletion approach and its inhibitory effects on cytochromes P450 (CYP) activities were evaluated using probe substrates, with IC(50) and the (K(i)) values were 1.2 microM (0.5 microM) and 1.3 microM (1.9 microM), respectively for CYP3A4/5 (midazolam) and CYP2C19 (mephenytoin). Similarly, beauvericin was also a potent inhibitor for CYP3A1/2 (IC(50): 1.3 microM) in RLM. Furthermore, the pharmacokinetics of beauvericin in the rat were studied after p.o administration alone and co-administration with ketoconazole, which indicated a pharmacodynamic function may play a role in the synergistic effect on antifungal activity.     

Further immunochemical and biocatalytic characterization of CYP1A1 from feral leaping mullet liver (Liza saliens) microsomes

CYP1A is known to play important roles in the metabolism, detoxification and bioactivation of carcinogens and other xenobiotics in animals including fish. In our laboratory, CYP1A1 was obtained in a highly purified form with a specific content of 15-17 nmol P450 per mg protein from liver microsomes of feral fish, leaping mullet (Liza saliens). Purified mullet CYP1A1 showed a very high substrate specificities for 7-ethoxyresorufin and 7-methoxyresorufin in a reconstituted system containing purified fish P450 reductase and lipid. In addition, effects of each individual components of the reconstituted system, i.e., CYP1A1 and P450 reductase on 7-methoxyresorufin O-demethylase (MROD) activity were studied. 7-ethoxyresorufin O-deethylase (EROD) activity was strongly inhibited by alpha-naphthoflavone (ANF). At 0.5 and 2.5 microM. ANF inhibited EROD activity by 90 and 98%, respectively. Mullet CYP1A1 did not catalyze monooxygenations of other substrates such as aniline, ethylmorphine, N-nitrosodimethylamine and p-nitrophenol. Antibodies produced against CYP1A1 orthologues in fish such as trout and scup showed strong cross-reactivity with the purified mullet CYP1A1. In addition, anti-L. saliens liver CYP1A1 produced in our laboratory inhibited both the EROD and MROD activities catalyzed by L. saliens liver microsomes but stronger inhibition was observed with EROD activity. On the other hand, anti-mullet CYP1A1 antibodies showed very weak cross-reactivity with two proteins (presumably CYP1A1 and CYP1A2) in 3MC-treated rat liver microsomes. Moreover, 3MC-treated rat liver microsomal EROD activity was weakly inhibited by the anti-L. saliens liver CYP1A1. These results strongly suggested that the purified mullet CYP1A1 is structurally, functionally and immunochemically similar to the CYP1A1 homologues purified from other teleost species but functionally and immunochemically distinct from mammalian CYP1A1.