Immunohistochemical localization and differential expression of cytochrome P450 3A27 in the gastrointestinal tract of rainbow trout.

In mammals the cytochrome P450 3A (CYP3A) subfamily isoforms are primarily expressed in liver and intestines with lesser amounts found in other tissues. The aim of this study was to examine the cellular localization and the expression pattern of CYP3A27 in the gastrointestinal tract (GI tract) of a freshwater teleost species, the rainbow trout (Oncorhynchus mykiss), a fish model used extensively for toxicological and carcinogenesis research. Using an avidin biotinylated enzyme complex and 3,3'-diaminobenzidine staining, strong cytoplasmic immunohistochemical staining was observed for CYP3A27 protein in hepatocytes and in enterocytes of the intestinal ceca and the proximal descending intestine when probed with a polyclonal antibody raised against rainbow trout P450 LMC5, a CYP3A protein. The intensity of epithelial staining decreased distally along the GI tract with faint staining observed in the epithelial cells examined near the vent. Western blot analysis was supportive of the immunohistochemistry results. Northern blot analysis also demonstrated that CYP3A27 mRNA was expressed along the entire GI tract. The major area of CYP3A27 mRNA expression was in the intestinal ceca, followed by the proximal descending intestine, at levels that were about three- to five-fold and two- to four-fold, respectively, greater than seen in the liver of the fish studied. Monooxygenase activities of intestinal ceca microsomes against testosterone and progesterone confirmed the presence of active CYP3A enzyme in this tissue. These results suggest that the intestine of rainbow trout may possesses substantial capacity for first-pass metabolism of xenobiotics by CYP3A27, which makes it an excellent model in which to study the consequence of such metabolism.     

Characterization of CYP1A1 and CYP1A3 gene expression in rainbow trout (Oncorhynchus mykiss)

The rainbow trout CYP1A1 and CYP1A3 genes share 96% amino acid identity and have similar enzymatic activity. The expression of CYP1A1 and 1A3 genes was investigated in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated rainbow trout tissues, sac fry and cell lines. Both CYP1A1 and CYP1A3 were induced by TCDD in all the tissues examined. While CYP1A3 gene was expressed constitutively at higher levels than CYP1A1 in trout intestine, preferential expression of CYP1A1 occurred in trout liver, heart, kidney and trout sac fry. In rainbow trout gonad (RTG)-2 and rainbow trout hepatoma (RTH)-149 cell lines, CYP1A1 was constitutively expressed and induced by exposure to TCDD, but CYP1A3 message was not detected, even after TCDD treatment. Quantitative analysis of CYP1A genes expression in rainbow trout liver revealed that TCDD induced CYP1A1 expression more than 50-fold and CYP1A3 RNA levels increased at least 100-fold over untreated fish. The cell- and tissue-specific expression indicates that these closely related CYP1A genes are independently regulated and that negative regulation may play a role in CYP1A3 gene expression.     

Cellular localization of CYP3A proteins in various tissues from pilot whale (Globicephala melas)

The in situ expression of cytochrome P450 3A- (CYP3A) like proteins in hepatic and extrahepatic tissues from a marine mammal, pilot whale (Globicephala melas), was investigated. Polyclonal antibodies (PAb) raised against either rat CYP3A1 or trout CYP3A27 both recognized a microsomal protein band in liver, lung, kidney and heart. The protein band observed in liver and lung had slightly lower molecular weight than that observed in kidney and heart, suggesting the existence of two CYP3A forms in pilot whale. Immunohistochemical analyses showed strong CYP3A-staining in hepatocytes, bile duct epithelial cells, bronchial epithelial cells, in primordial- and primary follicles and their surrounding zona glomerulosa. Moderate to strong CYP3A staining was seen in smooth muscle-like cells of large arteries and arterioles in all organs examined. Mild to moderate staining was evident in alveolar epithelial cells and in kidney tubular epithelial cells. Weak staining was seen in glomerular epithelial cells and in seminiferous tubular epithelial cells.     

Functional properties of a rainbow trout CYP3A27 expressed by recombinant baculovirus in insect cells.

Cytochrome p450 3A27 (CYP3A27) is highly expressed in liver and intestine of rainbow trout (Oncorhynchus mykiss). In many animal species, the intestine and liver are responsible for the first-pass metabolism of a wide range of xenobiotics. To help determine its physiological role, the catalytic capabilities of CYP3A27 protein were examined. An open reading frame of CYP3A27 in pFastBac donor plasmid was transferred to the baculovirus genome (bacmid DNA) through Tn7 site-specific transposition in DH10Bac competent cells. The CYP3A27 cDNA was positioned under the control of the polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus. The recombinant baculovirus containing a full-length CYP3A27 cDNA (Bv-3A27) was then transfected into Spodoptera frugiperda (Sf9) insect cells for overexpression of CYP3A27 protein. The expressed CYP3A27 protein (714 pmol/mg total protein) exhibited a maximum CO-reduced spectrum at 450 nm at 72 h postinfection after addition of 1 micro g/ml exogenous hemin. The expressed CYP3A27 protein comigrated with the purified trout LMC5 cytochrome p450 (p450) and was recognized by anti-p450 LMC5 IgG on Western blot analysis. The expressed CYP3A27 protein was reconstituted with human NADPH-cytochrome p450 reductase and cytochrome b(5). The reconstitution system showed catalytic activities for the 6 beta-, 2 beta-, and 16 beta-hydroxylation of testosterone at 1.428, 0.043, 0.034 nmol/min/nmol CYP3A27, respectively, and the dehydrogenation of nifedipine at 50 pmol/min/nmol CYP3A27. The present results demonstrated that the baculovirus system is useful for the production of the functional aquatic CYP3A form and that CYP3A27 has the capability to metabolize steroid hormone as reported for mammalian CYP3A forms.     

CYP3A in horse intestines

The intestinal enterocytes provide the initial site for cytochrome P450 (CYP)-mediated metabolism of orally absorbed xenobiotics. In man and some animal species, the CYP3A subfamily is highly expressed in the intestines and considered to be important in the first-pass metabolism of drugs and other xenobiotics. The aim of the present study was to investigate the mRNA expression, immunohistochemical localization and catalytic activity of CYP3A in the intestines of horse. Real-time RT-PCR analyses showed that the highest CYP3A mRNA expression was present in the duodenum with a decreasing level towards jejunum, ileum, cecum, and colon. The CYP3A mRNA expression in the liver was similar as in the anterior part of the jejunum, but about 4.5 times lower than in the anterior part of the duodenum. Immunohistochemistry showed CYP3A immunoreactivity in the cytoplasm of the enterocytes, which decreased distally along the intestinal tract. CYP3A-dependent metabolic activity rose slightly from the anterior to the distal part of the duodenum and the anterior part of the jejunum and then declined to the middle and distal parts of the jejunum and the ileum, cecum, and colon. Our results suggest that CYP3A in the small intestine plays a major role in first-pass metabolism and may affect bioavailability and therapeutic efficiency of some orally administrated drugs in horse.     

Properties and regional expression of a CYP3A-like protein in channel catfish intestine

Biotransformation in the intestine may influence the bioavailability and toxicity of ingested xenobiotics. The objective of this study was to examine the expression and catalytic properties of a constitutive cytochrome P450 (CYP) 3A-like protein along the intestine of channel catfish, Ictalurus punctatus. Fish were maintained on commercial chow or nutritionally complete semi-purified diets. Polyclonal antibodies generated against rainbow trout CYP3A proteins reacted strongly with catfish washed intestinal microsomes on Western blots showing a major protein band with MW of 59 kDa. In catfish maintained on a standard chow diet, the expression of this protein was higher in the proximal segment (0.101 +/- 0.031 units/mg protein, mean +/- S.D., n = 4) than in the distal part (0.032 +/- 0.023 units/mg protein). Microsomal testosterone 6beta-hydroxylation activity was monitored as the catalytic indicator of CYP3A, and was higher in proximal than distal catfish intestine (263 +/- 80.3 and 88.6 +/- 15.6 pmol/min/mg protein for proximal and distal, respectively, mean +/- S.D., n = 4). CYP3A protein levels and testosterone 6beta-hydroxylation activities were lower in microsomes from the proximal segment of intestine from catfish maintained on a semi-purified diet, compared with commercial chow, but again the proximal intestine had higher CYP3A and 6beta-hydroxylase activities than distal intestine. Testosterone 6beta-hydroxylase activities in all samples correlated with the CYP3A protein levels, r2 = 0.8. Testosterone 6beta-hydroxylation was inhibited by specific CYP3A inhibitors, ketoconazole (IC50 = 0.02 microM) and erythromycin (IC50 = 41 microM), as well as general CYP inhibitors, metyrapone (IC50 = 2.8 microM) and SKF-525A (IC50 = 25 microM). There was evidence for the involvement of CYP3A in the mono-oxygenation of benzo(a)pyrene and of (-)-benzo(a)pyrene-7,8-dihydrodiol in intestinal microsomes from catfish maintained on the semi-purified diet. Mono-oxygenation of both substrates was increased in a concentration-dependent manner by in vitro addition of alpha-naphthoflavone. Benzo(a)pyrene hydroxylase activities were higher in proximal than in distal intestine; 3.72 +/- 0.77 pmol/min/mg protein, mean +/- S.D., n = 5 and 1.45 +/- 0.42 in these respective segments. The results of this study strongly suggest that CYP3A is important in the first pass metabolism of dietary xenobiotics in untreated fish.     

Ketoconazole, an antifungal imidazole, increases the sensitivity of rainbow trout to 17alpha-ethynylestradiol exposure

This study focuses on effects of two classes of xenobiotics, azole fungicides and xenoestrogens, both of which have been detected in the aquatic environment. We hypothesize that azoles and estrogenic compounds are metabolized by cytochrome P450 (CYP) enzymes, and in particular CYP1A and CYP3A, to more readily excreted metabolites. We exposed rainbow trout (Oncorhynchus mykiss) to two different pharmaceutical representatives of theses two classes, such as the imidazole ketoconazole and the synthetic estrogen analogue, 17alpha-ethynylestradiol (EE(2)). Juvenile rainbow trout were i.p. injected with a single low dose of EE(2) (2.5 microg/kg), alone or in combination with ketoconazole (100mg/kg). Hepatic microsomal CYP1A and CYP3A protein expressions were analyzed in Western blots using polyclonal antibodies (PAb) and enhanced cheminoluminescence. CYP1A activities were analyzed using the ethoxyresorufin-O-deethylase (EROD) assay and CYP3A activities were analyzed using the benzyloxy-4-[trifluoromethyl]-coumarin-O-debenzyloxylase (BFCOD) assay. Plasma vitellogenin (vtg) and sex steroid hormones (i.e. 17beta-estradiol, testosterone and 11-keto-testosterone) were analyzed using commercially available ELISA-kits. The vtg mRNA expression was analyzed using quantitative (Q)-PCR. The dose of EE(2) selected had little or no effect on the estrogen receptor (ER) mediated vtg induction. However, in combination with ketoconazole this threshold-dose of EE(2) resulted in significantly elevated plasma vtg levels, 6 days post injection. Exposure to ketoconazole resulted in up to nine-fold induction of CYP1A after 3 days. However, this nine-fold induction was not reflected on the CYP1A catalytic activity, where exposure to ketoconazole resulted only in a two-fold increase in activity. Ketoconazole increased CYP3A protein levels 1.5-fold and decreased BFCOD activities by 80% at days 3 and 6. Treatment with ketoconazole and EE(2) alone and in combination had no significant effect on sex steroid hormones, compared to vehicle-treated fish. This study demonstrates that exposure to ketoconazole compromises the function of key enzymes involved in metabolic clearance of xenobiotics and steroids, and increases the sensitivity to EE(2) exposure in juvenile rainbow trout.     

Gill EROD in monitoring of CYP1A inducers in fish: a study in rainbow trout (Oncorhynchus mykiss) caged in Stockholm and Uppsala waters

The gill filament 7-ethoxyresorufin O-deethylase (EROD) assay was evaluated as a monitoring tool for waterborne cytochrome P4501A (CYP1A) inducers using rainbow trout (Oncorhynchus mykiss) caged in urban area waters in Sweden. To compare the CYP1A induction response in different tissues, EROD activity was also analyzed in liver and kidney microsomes. Immunohistochemistry was used to localize CYP1A protein in gill and kidney. In two separate experiments fish were caged at sites with fairly high expected polyaromatic hydrocarbon (PAH) contamination. In the first experiment, gill EROD activities were analyzed in fish exposed for 1-21 days in a river running through Uppsala. The reference site was upstream of Uppsala. In the second, gill, liver and kidney EROD activities were analyzed in fish exposed for 1-5 days in fresh or brackish waters of Stockholm and in a reference lake 60km north of Stockholm. Fish exposed for 5 days followed by 2 days of recovery in tap water in the laboratory were also examined. The gill consistently showed a higher EROD induction compared with the liver and the kidney. After 1 day of caging, gill EROD activity was markedly induced (6-17-fold) at all sites examined. Induction in gill was pronounced (5-7-fold) also in fish caged at the reference sites. In the 21-day exposure study gill EROD activity remained highly induced throughout the experiment (26-fold at most) and the induced CYP1A protein was exclusively confined to the gill secondary lamellae. In the 5-day exposure experiment, EROD activity peaked after 1 day and then declined in both gill and liver, while CYP1A immunostaining in the gill remained intense over the 5-day period. In the kidney, CYP1A staining was weak or absent. We conclude that gill EROD activity is a more sensitive biomarker of exposure to waterborne CYP1A inducers than EROD activity in liver and kidney.     

Using real-time quantitative TaqMan RT-PCR to evaluate the role of dexamethasone in gene regulation of rat P-glycoproteins mdr1a/1b and cytochrome P450 3A1/2

Cytochromes P450 (CYPs) and p-glycoproteins (Pgps) are believed to play important roles in drug absorption, metabolism, and elimination. Numerous drugs and environmental chemicals can modulate expression of these two classes of genes in different species. The present study investigated the effect of dexamethasone (Dex) on gene expression on both message and protein levels of mdr1a, mdr1b, CYP3A1, and CYP3A2 in small intestine, colon, liver, kidney, and brain microvessels of the rats treated orally with Dex at 1 or 20 mg/kg/day for 3 days. The basal expression of mdr1a mRNA was highest in the brain microvessels followed by colon, small intestine, liver, and kidney, and mdr1b mRNA was highest in the brain microvessels followed by kidney, liver, colon, and small intestine. After Dex treatment, mdr1a mRNA was increased by 5.5- and 10.7-fold in the small intestine, decreased extensively by 85-90% in the liver, and showed little or no change in the colon, kidney, and brain microvessels compared to the control rats. A similar pattern was observed for mdr1b mRNA. CYP3A1 mRNA was increased in all tissues examined. CYP3A2 mRNA was not significantly changed with the exception that at 20 mg/kg CYP3A2 mRNA was increased 5- and 30-fold in the colon and kidney. In general, Western blot analyses were consistent with mRNA changes. CYP3A protein expression was increased in all tissues examined. The disparity of the impact of Dex on the CYP 3A and Pgp expression in these studies suggest that the regulation of Pgp expression is very complex and is difficult to predict solely based on the PXR response to xenobiotics.     

Identification of a putative calcium-binding protein as a dioxin-responsive gene in zebrafish and rainbow trout

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin) is a widespread environmental contaminant that causes multiple effects in vertebrates. TCDD elicits its toxicity through aryl hydrocarbon receptor (AhR)-mediated modulation of gene regulation, increasing intracellular free calcium, and inducing calcium-mediated apoptosis in cell culture. Two TCDD-responsive cDNAs, which encode putative calcium-binding proteins, have been isolated from zebrafish and rainbow trout. The zebrafish and rainbow trout sequences are 88% similar to each other at the amino acid level and are orthologs of the human S100A4 calcium-binding protein. In zebrafish liver cell culture, treatment with TCDD increases S100A4a mRNA abundance. In juvenile rainbow trout, S100A4 mRNA was constitutively expressed in the heart, kidney, intestine, and spleen, but not in the liver. Exposure to TCDD significantly increased rainbow trout S100A4 mRNA abundance in the rainbow trout kidney. Taken together, these findings demonstrate in zebrafish and rainbow trout that dioxin increases expression of this EF-hand calcium-binding protein gene in a tissue-dependent fashion. However, demonstration that the encoded S100A4 proteins actually bind calcium and play a role in dioxin toxicity will require further study.     

Effects of salinity on aldicarb toxicity in juvenile rainbow trout (Oncorhynchus mykiss) and striped bass (Morone saxatilis x chrysops).

Fluctuations in several environmental variables, such as salinity, can influence the interactions between organisms and pollutants in aquatic organisms, and, therefore, affect the toxicity of xenobiotics. In this study, after 2 species of fish, rainbow trout (Oncorhynchus mykiss) and hybrid striped bass (Morone saxatilis x chrysops) were acclimated to 4 salinity regimens of 1.5, 7, 14, and 21 ppt for 1 week and then exposed to 0.5 mg/l aldicarb. Mortality, brain, and muscle cholinesterase levels were measured after 96 h. Rates of (14)C-aldicarb sulfoxide formation were determined in kidney (trout only), liver, and gill microsomes from each species acclimated to the 4 salinity regimens. Salinity significantly enhanced aldicarb toxicity, cholinesterase inhibition, and (14)C-aldicarb sulfoxide formation in rainbow trout but not in striped bass. In vitro incubations with (14)C-aldicarb and the cytochrome P450 (CYP) inhibitor, N-benzylimidazole, did not significantly alter aldicarb sulfoxide formation in tissue microsomes from either species of fish, indicating CYP did not contribute to aldicarb sulfoxidation. Salinity increased flavin-containing monooxygenase (FMO) mRNA expression and catalytic activities in microsomes of liver, gill, and kidney of rainbow trout, which was consistent with the salinity-induced enhancement of aldicarb toxicity. Salinity did not alter FMO mRNA expression and catalytic activities in striped bass, which was also consistent with the lack of an effect of salinity on aldicarb toxicity in this species. These results suggest that salinity-mediated enhancement of aldicarb toxicity is species-dependent, and at least partially due to the salinity-related upregulation of FMOs, which, in turn, increases the bioactivation of aldicarb to aldicarb sulfoxide, which is a more potent inhibitor of cholinesterase than aldicarb.     

Expression of CYP3A4 mRNA is correlated with CYP3A4 protein level and metabolic activity in human liver

A relatively large amount of human liver tissue was required to determine the exact activity of human hepatic CYP3A. Although, the quantity of available human liver tissue samples is limited. We measured levels of CYP3A4 mRNA by RT-PCR with a radiolabeled primer specific for CYP3A4 and compared mRNA expression with CYP3A4 protein level and metabolic activity in liver. The level of CYP3A4 mRNA was correlated with the levels of CYP3A4 protein and activity. Our results suggest that CYP3A4 protein and activity levels can be predicted from CYP3A4 mRNA levels determined by RT-PCR and using a very small amount of liver tissue.