Testosterone metabolism by microsomal cytochrome P-450 in liver of rats treated with some inducers

1. The stereoselective hydroxylation of testosterone by microsomal cytochrome P-450 and the changes in level of components participated in the microsomal electron transport system were observed in the microsomes induced unique P-450 isozymes. 2. Flavone- and hesperetin-inducible P-450 catalyzed the hydroxylation of testosterone more effectively than other chemicals-inducible ones. 3. The P-450 in all the microsomal preparations tested most rapidly oxidized testosterone to 6 beta-monohydroxy form. 4. Particularly, MC- and BNF-inducible P-450 showed high stereoselectivity on C6-position of testosterone, and PB-, flavone- and hesperetin-inducible one showed that on C2-position of this compound, respectively. 5. This specificity of two flavonoid-inducible P-450 for the formation of 2 alpha- and 2 beta-epimer of monohydroxytestosterone was opposite to each other. 6. The content of P-450 and the activity of NADPH-cytochrome P-450 reductase were high in PB-, MC- and BNF-microsomes, whereas NADH-cytochrome b5 reductase activity was high in two flavonoid-microsomes and the content of cytochrome b5 was not changed except the PB-treated rats. 7. It is suggested that the increasing activities of testosterone hydroxylases in flavonoid-microsomes seems to be closely related to NADH-cytochrome b5 reductase.     

Impact of deltamethrin exposure on mRNA expression levels of metallothionein A, B and cytochrome P450 1A in rainbow trout muscles

Metallothioneins (MT) are widely utilized to identify specific responses to heavy metal pollution. In addition, there is evidence demonstrating that in vertebrates MT synthesis is stimulated by different endogenous and exogenous agents in particular compounds leading to production of ROS. Also, cytochrome P450 1A can enhance the generation of ROS. On this basis, MT and CYP 1A induction can be considered as biomarkers of oxidative stress. In the current study, we examined the influences of pesticide administration on the expression of MT-A, MT-B and CYP 1A. For this purpose, we produced muscle metallothionein-A, metallothionein-B and cytochrome P450 1A cDNAs and used quantitative RT-PCR to assay mRNAs in rainbow trout exposed to acute and long-term deltamethrin administration. We observed that deltamethrin exposure significantly (p<0.05) increased the expression levels of Cyp1A, MT-A and MT-B in a time dependent manner. Results of our study contributes to the identification of inducers of such biomarkers in addition to well known agents.     

Interaction between cytochrome P450 and other drug-metabolizing enzymes: evidence for an association of CYP1A1 with microsomal epoxide hydrolase and UDP-glucuronosyltransferase

Protein-protein interactions between cytochrome P450 (P450) and other drug-metabolizing enzymes were studied by affinity chromatography using CYP1A1-, glycine-, and bovine serum albumin (BSA)-conjugated Sepharose 4B columns. Sodium cholate-solubilized microsomes from phenobarbital-treated rat liver were applied to the columns and the material eluted with buffer containing NaCl was analyzed by immunoblotting. Microsomal epoxide hydrolase (mEH) and UDP-glucuronosyltransferases (UGTs), as well as NADPH-P450 reductase, were efficiently trapped by the CYP1A1 column. Glycine and BSA columns exhibited no ability to retain these proteins. Protein disulfide isomerase and calnexin, non-drug-metabolizing enzymes expressed in the endoplasmic reticulum, were unable to associate with the CYP1A1 column. These results suggest that CYP1A1 interacts with mEH and UGT to facilitate a series of multistep drug metabolic conversions.     

Differential inhibition of CYP17A1 and CYP21A2 activities by the P450 oxidoreductase mutant A287P

P450 oxidoreductase (POR) has a pivotal role in facilitating electron transfer from nicotinamide adenine dinucleotide phosphate to microsomal cytochrome P450 (CYP) enzymes, including the steroidogenic enzymes CYP17A1 and CYP21A2. Mutations in POR have been shown recently to cause congenital adrenal hyperplasia with apparent combined CYP17A1 and CYP21A2 deficiency that comprises a variable clinical phenotype, including glucocorticoid deficiency, ambiguous genitalia, and craniofacial malformations. To dissect structure-function relationships potentially explaining this phenotypic diversity, we investigated whether specific POR mutations have differential effects on CYP17A1 and CYP21A2. We compared the impact of missense mutations encoding for single amino acid changes in three distinct regions of the POR molecule: 1), Y181D and H628P close to the central electron transfer area, 2) S244C located within the hinge close to the flavin adenine dinucleotide and flavin mononucleotide domains of POR, and 3) A287P that is clearly distant from the two other regions. Functional analysis using a yeast microsomal assay with coexpression of human CYP17A1 or CYP21A2 with wild-type or mutant human POR revealed equivalent decreases in CYP17A1 and CYP21A2 activities by Y181D, H628P, and S244C. In contrast, A287P had a differential inhibitory effect, with decreased catalytic efficiency (Vmax/Km) for CYP17A1, whereas CYP21A2 retained near normal activity. In vivo analysis of urinary steroid excretion by gas chromatography/mass spectrometry in 11 patients with POR mutations showed that A287P homozygous patients had the highest corticosterone/cortisol metabolite ratios, further indicative of preferential inhibition of CYP17A1. These findings provide novel mechanistic insights into the redox regulation of human steroidogenesis. Differential interaction of POR with electron-accepting CYP enzymes may explain the phenotypic variability in POR deficiency, with additional implications for hepatic drug metabolism by POR-dependant CYP enzymes.     

Erythrocyte antioxidative enzyme activities and lipid peroxidation levels in patients with cutaneous leishmaniasis

Although cutaneous leishmaniasis (CL) is a local infection, the cellular immune response against the disease is systemic, and reactive oxygen intermediates (ROIs) are an important part of cellular immune response involved in killing the parasite. However, whether these intermediates cause oxidative damage in host is unknown. In this study, the metabolism of ROIs were investigated in patients with CL, and compared with healthy subjects. Erythrocyte lipid peroxidation was determined, as an index of oxidative damage, by measurement of malondialdehyde (MDA) levels. Erythrocyte MDA levels and SOD activities were found to be significantly higher in CL patients than in control subjects (p < 0.01 and p < 0.01, respectively). However, CAT and GSH-Px activities were significantly lower in the CL group (p < 0.05 and p < 0.01, respectively). There was a tendency to decreased hemoglobin (Hb) levels, but values did not reach statistical significance. These findings suggest that changes in antioxidant enzyme activities may amplify the leishmanicidal effect in patients with CL. However, these changes may not only cause the killing of parasite but also may cause oxidative damage in the other cells or tissues.     

Cytochrome P450 1A1 (CYP1A1) in blood lymphocytes evidence for catalytic activity and mRNA expression.

Studies initiated to characterise the catalytic activity and expression of CYP1A1 in rat blood lymphocytes revealed significant activity of 7-ethoxyresorufin-O-deethylase (EROD) in rat blood lymphocytes. Pretreatment with 3-methylcholanthrene (MC) and beta-naphthoflavone (NF) resulted in significant induction in the activity of lymphocyte EROD suggesting that like the liver enzyme, EROD activity in lymphocytes is inducible and is mediated by the MC inducible isoenzymes of P450. The increase in the activity of EROD was associated with a significant increase in the apparent Vmax and affinity of the substrate towards EROD. That this increase in the activity of EROD could be primarily due to the increase in the expression of CYP1A1 isoenzymes was demonstrated by RT-PCR and western immunoblotting studies indicating an increase in the expression of CYP1A1 in blood lymphocytes after MC pretreatment. Significant inhibition in the EROD activity of MC induced lymphocyte by anti-CYP1A1/1A2 and alpha-naphthoflavone further provided evidence that the CYP1A1/1A2 isoenzymes are involved in the activity of EROD in blood lymphocytes. The data indicating similarities in the regulation of CYP1A1 in blood lymphocytes with the liver isoenzyme suggests that factors which may affect expression of CYP1A1 in liver may also affect expression in blood lymphocytes and that blood lymphocytes could be used as a surrogates for studying hepatic expression of the xenobiotic metabolising enzymes.     

Cytochrome P450 2E1 dependent catalytic activity and lipid peroxidation in rat blood lymphocytes

To investigate the similarities in the catalytic activity of blood lymphocyte P450 2E1 in blood lymphocyte with the liver isoenzyme, NADPH dependent lipid peroxidation and activity of N-nitrosodimethyamine demethylase (NDMA-d) was studied in rat blood lymphocytes. Blood lymphocytes were found to catalyse NADPH dependent (basal) lipid peroxidation and demethylation of N-nitrosodimethylamine (NDMA). Pretreatment with ethanol or pyrazole or acetone resulted in significant increase in the NADPH dependent lipid peroxidation and the activity of NDMA-d in blood lymphocytes and liver microsomes. In vitro addition of CCl(4) to the blood lymphocytes isolated from control or ethanol pretreated rats resulted in an increase in the NADPH dependent lipid peroxidation. Significant inhibition of the basal and CCl(4) supported NADPH dependent lipid peroxidation and NDMA-d activity in blood lymphocytes isolated from control or ethanol pretreated rats by dimethyl formamide or dimethyl sulfoxide or hexane, solvents known to inhibit P450 2E1 catalysed reactions in liver and anti- P450 2E1, have indicated the role of P450 2E1 in the NADPH dependent lipid peroxidation in rat blood lymphocytes. The data indicating similarities in the NADPH dependent lipid peroxidation and NDMA-d activity in blood lymphocyte with the liver microsome have provided evidence that blood lymphocyte P450 2E1 could be used as a surrogate to monitor and predict hepatic levels of the enzyme.     

Ornithine alpha-ketoglutarate modulates the levels of antioxidants and lipid peroxidation products in ammonium acetate treated rats

The effects of ornithine alpha-ketoglutarate (OKG) on ammonium acetate induced hepatotoxicity were studied in experimental rats. The levels of urea, non-protein nitrogen and thiobarbituric acid reactive substances were significantly increased in ammonium acetate treated rats; but these levels were significantly decreased in ammonium acetate-OKG treated rats. Similar patterns were observed in the levels of free fatty acids, triglycerides and phospholipids. Furthermore, non-enzymatic (reduced glutathione) and enzymatic (glutathione peroxidase, superoxide dismutase and catalase) antioxidants were significantly decreased in ammonium acetate treated rats, when compared with control and were significantly increased in ammonium acetate-OKG treated rats compared to ammonium acetate treatment alone.     

Interleukin-1 (IL-1) depresses cytochrome P450 levels and activities in mice

Endotoxin depresses cytochrome P450 levels when injected into animals. The purpose of this study was to determine whether endotoxin itself, or monokine(s) released in response to endotoxin administration are responsible for this effect. Cytochrome P450 levels and drug metabolizing activities were measured in endotoxin resistant C3H/HeJ mice 24h after single intraperitoneal injections of either lipopolysaccharide (LPS), a semipurified murine monokine preparation containing interleukin-1 (IL-1), or murine recombinant IL-1. In endotoxin sensitive C3H/HeN mice, LPS (0.5 mg/Kg) decreased total cytochrome P450 levels, benzphetamine demethylase activities, and ethoxyresorufin-0-deethylase activities. This dose of LPS did not alter cytochrome P450 levels or activities in the C3H/HeJ mice. However, after injection of the semipurified monokine preparation or the recombinant IL-1, there were significant decreases in cytochrome P450 levels and activities similar to the decreases observed with LPS in the C3H/HeN mice. These findings suggest that the alterations in hepatic cytochrome P450 seen with endotoxin injection are mediated, at least in part, by IL-1.     

Features of the retinal environment which affect the activities and product profile of cholesterol-metabolizing cytochromes P450 CYP27A1 and CYP11A1

The retina is the sensory organ in the back of the eye which absorbs and converts light to electrochemical impulses transferred to the brain. Herein, we studied how retinal environment affects enzyme-mediated cholesterol removal. We focused on two mitochondrial cytochrome P450 enzymes, CYPs 27A1 and 11A1, which catalyze the first steps in metabolism of cholesterol in the retina and other tissues. Phospholipids (PL) from mitochondria of bovine neural retina, retinal pigment epithelium, liver and adrenal cortex were isolated and compared for the effect on kinetic properties of purified recombinant CYPs in the reconstituted system in vitro. The four studied tissues were also evaluated for the mitochondrial PL and cholesterol content and levels of CYPs 27A1, 11A1 and their redox partners. The data obtained were used for modeling the retinal environment in the in vitro enzyme assays in which we detected the P450 metabolites, 22R-hydroxycholesterol and 5-cholestenoic acid, unexpectedly found by us in the retina in our previous studies. The effect of the by-product of the visual cycle pyridinium bis-retinoid A2E on kinetics of CYP27A1-mediated cholesterol metabolism was also investigated. The results provide insight into the retina's regulation of the enzyme-mediated cholesterol removal.     

Diverse action of acrylamide on cytochrome P450 and glutathione S-transferase isozyme activities,mRNA levels and protein levels in human hepatocarcinoma cells

Humans are exposed to acrylamide in their diet and cigarette smoke. Acrylamide is metabolized into glycidamide by CYP2E1. However, very few studies regarding the effects of acrylamide on cytochrome P450 and Glutathione S-Transferase (GST) isozymes have been pursued. The aim of this study is to elucidate the effects of acrylamide on cytochrome P450 and GST isozymes in HepG2 cell line. Treatment with 1.25 and 2.5 mM acrylamide caused 9.5- and 3.7-fold increases and 4.0- and 3.3-fold increases in CYP1A-associated ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) activities, respectively. These increases were consistent with increases in mRNA and protein levels of these isozymes. Similarly, CYP2E1-associated aniline 4-hydroxylase (ANH) activity, protein levels, and mRNA levels increased 2.1- and 2.6-fold, 2.4- and 3.2-fold, and 1.4- and 1.9-fold following 1.25 and 2.5 mM acrylamide treatments, respectively. In addition, GST-mu activity was increased 2.4- and 5.1-fold by acrylamide. Moreover, GST-mu mRNA and protein levels increased twofold as a result of acrylamide treatment. In contrast, GST-pi protein and mRNA levels decreased significantly. In conclusion, human cell exposure to acrylamide causes an increase in the levels of carcinogenicity and toxicity and a disturbance in drug metabolism, possibly due to complex effects on P450 and GST isozymes.     

A second CYP26 P450 in humans and zebrafish: CYP26B1.

A new P450 gene has been found in humans. It has 44% sequence identity to CYP26A1 from human and mouse, which places it in a new subfamily, CYP26B. There is only one human EST from a retinal library (AA012833) that matches the coding region. No homologous ESTs are found in mouse. A zebrafish EST AI721901 shows 68% identity to the human protein. This zebrafish EST is only 41% identical to the zebrafish CYP26A1 protein sequence, so it represents the homolog of the human CYP26B1 sequence. It is not known if this gene product will act on all-trans-retinoic acid like the CYP26A1 protein or if it might hydroxylate the 9-cis- or 13-cis-retinoic acid isoforms not recognized by CYP26A1. The importance of the CYP26A1 P450 in mouse and zebrafish development flags the CYP26B1 gene as a potential developmental gene.     

Induction of cytochrome P450 (CYP)1A1, CYP1A2, and CYP3A4 but not of CYP2C9, CYP2C19, multidrug resistance (MDR-1) and multidrug resistance associated protein (MRP-1) by prototypical inducers in human hepatocytes

Human hepatocytes cultured serum-free for up to 6 weeks were used to study expression and induction of enzymes and membrane transport proteins involved in drug metabolism. Phase I drug metabolizing enzymes cytochrome P450 (CYP)1A1, CYP1A2, CYP2C9, CYP2C19, CYP2E1, and CYP3A4 were detected by Western blot analyses and, when appropriate, by enzymatic assays for ethoxyresorufin-O-deethylase(EROD)-activity and testosterone-6beta-hydroxylase(T6H)-activity. Expression of the membrane transporter multi-drug resistance protein (P-glycoprotein, MDR-1), multidrug resistance-associated protein (MRP-1), and lung-resistance protein (LRP) was maintained during the culture as detected by RT-PCR and Western blot analyses. Model inducers like rifampicin, phenobarbital, or 3-methylcholanthrene and beta-naphtoflavone were able to induce CYP1A or CYP3A4 as well as EROD or T6H activities for up to 30 days. CYP2C9, CYP2C19 and CYP2E1 expression was maintained but not inducible for 48 days. Also, rifampicin and phenobarbital were unable to increase MDR-1 and MRP-1 protein levels significantly.     

A possible role of cAMP dependent phosphorylation of hepatic microsomal cytochrome P450: a mechanism to increase lipid peroxidation in response to hormone

Enzymatic lipid peroxidation in hepatocytes is believed to involve cytochrome P450. cAMP dependent phosphorylation of cytochrome P450 was found to increase the NADPH dependent production of malondialdehyde (lipid peroxidation) by about 30%. The cytochrome P450 inhibitor cyanide abolished this activity. The presence of spermine decreased the cytochrome P450 dependent lipid peroxidation in non-phosphorylated microsomes, phosphorylation partially reversed this effect. Thus, phosphorylation of cytochrome P450 and the associated increased lipid peroxidation may be a hormone dependent response to pathological conditions e.g. stress Phosphorylation was observed to subtly alter other properties of cytochrome P450. The rate of 7-ethoxycoumarin deethylase activity was reduced and the microwave power required to saturate the EPR spectrum of the low spin cytochrome P450 was decreased. It is hypothesized that phosphorylation of cytochrome P450 alters the interaction between the components of the cytochrome P450 system, which may enhance production of free radical species, initiating lipid peroxidation.     

Heme oxygenase-1 and NADPH cytochrome P450 reductase expression in experimental allergic encephalomyelitis: an expanded view of the stress response

Oxidative stress is implicated in the pathogenesis of experimental allergic encephalomyelitis (EAE), a model for multiple sclerosis. Heme oxygenase-1 (HO-1) is a heat shock protein induced by oxidative stress. HO-1 metabolizes the pro-oxidant heme to the antioxidant biliverdin and CO. HO-1 requires electrons, donated by NADPH cytochrome P450 reductase (henceforth, reductase), for catalytic activity. EAE was induced with a peptide of proteolipid protein in SJL mice, and the expression of HO-1 and reductase in the hindbrain was analyzed. HO-1 protein levels were significantly increased in EAE animals compared with control mice. HO-1 expression was present in ameboid macrophages, reactive microglia, and astrocytes in white matter tracks. Bergmann glia and ameboid macrophages also were occasionally stained in the molecular layer of the cerebellum. Unlike HO-1, reductase protein levels decreased with disease severity. HO-1 and reductase were associated with each other in endoplasmic reticulum micelles, suggesting that the decrease in reductase does not interfere with its association with HO-1. In cells that express HO-1, the association of reductase with HO-1 should competitively inhibit the interaction of reductase with cytochrome P450 isozymes and thereby limit free radical production as the latter two enzymes act cooperatively to produce superoxide. The increase in HO-1 together with the decrease in reductase may be part of a common defense mechanism attempting to minimize tissue damage in several neurological conditions.     

Genotype frequencies of polymorphic GSTM1, GSTT1, and cytochrome P450 CYP1A1 in Mexicans

The genotype frequencies of three metabolic polymorphisms were determined in a sample of a typical community in central Mexico. CYP1A1*3, GSTM1, and GSTT1 polymorphisms were studied in 150 donors born in Mexico and with Mexican ascendants; with respect to ethnicity the subjects can be considered Mestizos. PCR reactions were used to amplify specific fragments of the selected genes from genomic DNA. An unexpected 56.7% frequency of the CYP1A1*3 allele (which depends on the presence of a Val residue in the 462 position of the enzyme, instead of Ile) was found, the highest described for open populations of different ethnic origins (i.e., Caucasian, Asian, African, or African American). The GSTM1 null genotype was found with a frequency of 42.6%, which is not different from other ethnicities, whereas the GSTT1 null genotype had a frequency of 9.3%, one of the lowest described for any ethnic group but comparable to the frequency found in India (9.7%). The frequency of the combined genotype CYP1A1*3/*3 and the GSTM1 null allele is one of the highest observed to date (or perhaps the highest): 13.7% among all the ethnicities studied, including Caucasians and Asians, whereas the combination of CYP1A1*3/*3 with the GSTT1 null allele reached only 2.8%. The GSTM1 null allele combined with the GSTT1 null allele, on the other hand, has one of the lowest frequencies described, 4.24%, comparable to the frequencies found in African Americans and Indians. Finally, the combined CYP1A1*3/*3, GSTM1 null allele, and GSTT1 null allele genotype could not be found in the sample studied; it is assumed that the frequency of carriers of these combined genotypes is less than 1%. CYP1A1*3 and CYP1A1*2 polymorphisms were also evaluated in 50 residents in a community of northern Mexico; the CYP1A1*3 frequency was 54%, similar to that found in the other community studied, and the CYP1A1*2 frequency was 40%, which is high compared to Caucasians and Asians but comparable to the frequency found in Japanese and lower than the frequency found in Mapuche Indians. Haplotype frequencies for these CYP1A1 polymorphisms were estimated, and a linkage disequilibrium value (D) of 0.137 was calculated.     

Age-related changes in the mRNA levels of CYP1A1, CYP2B1/2 and CYP3A1 isoforms in rat small intestine

It has been established beyond doubt that, as well as the liver, the small intestine is an important site of first-pass metabolism of numerous drugs, food components and toxic xenobiotics. However, there is not much information available about age-dependent changes of intestinal biotransformation pathways. In the present paper, we evaluated the relationships between intestinal cytochrome P450 complex activity and the age of animals. The study was carried out on male Sprague–Dawley rats (n = 5) from 5 age series: 0.5-, 2-, 4-, 20-, and 28 months old. Animals at every age series were divided into 4 groups: control and three groups of rats treated with the CYP450 specific inducers: phenobarbital, β-naphtoflavone and dexamethasone, respectively. RNA was isolated from intestinal mucosa, and then standard RT-PCR was used for the analysis of CYP1A1, CYP2B1/2 and CYP3A1 mRNA expression. Additionally, the activities of NADPH-cytochrome P450 and NADH-cytochrome b₅ reductases in the microsomal fraction were biochemically estimated. The constitutive intestinal CYP1A1 mRNA expression changes during maturation and aging. Inducibility of CYP1A1 gene was evident in intestinal mucosa at 2-, 4- and 20-month-old rats. A similar pattern of changes was observed for CYP2B1/2 isoforms. CYP3A1 mRNA expression was not detected in small intestine of 2-week-old rats. In matured rats, constitutive intestinal CYP3A1 expression was low, although after induction, significant increases in CYP3A1 mRNA amount were noted in aged individuals. Intestinal activity of both analyzed reductases was lowest in immature rats and highest in 28-month-old animals. In conclusion, the activity of cytochrome P450 complex in rat small intestine was not decreased by the aging processes, so the high rate of oxidative metabolic reactions in intestinal mucosa can be maintained till the advanced life stage.     

Suppression in the expression of a male-specific cytochrome P450, P450-male: difference in the effect of chemical inducers on P450-male mRNA and protein in rat livers

Hypophysectomy of male adult rats caused a 70% decrease in the hepatic level of mRNA hybridized to two specific oligonucleotide probes for the sequence of coding and 3'-noncoding regions of P450(M-1) (H. Yoshioka et al., (1987) J. Biol. Chem. 262, 1706-1711), which corresponds to P450-male. Treatment of hypophysectomized male and female rats with subcutaneous injection of human growth hormone twice a day for 7 days increased the mRNA to a level similar to that of normal male rats. In contrast, the mRNA was decreased by treatment with continuous infusion. These results correlated well with those on the amounts of P450-male protein, indicating that growth hormone regulates the hepatic level of P450-male protein mainly by acting at the pretranslational step. Treatment of adult male rats with phenobarbital (PB), dexamethasone (Dex), or 3-methylcholanthrene (MC) decreased the content of P450-male protein by 68, 36, and 46%, respectively. The content of P450-male protein was also decreased to 65% in Dex-treated hypophysectomized male rats, but was not changed by treatment of hypophysectomized male rats with PB or MC, suggesting that PB and MC decrease P450-male protein through a pituitary growth hormone-mediated process. However, the level of mRNA hybridizable to the P450-male oligonucleotide probe was not decreased, but rather it increased in PB- or Dex-treated hypophysectomized male rats. A similar inconsistent change in protein and mRNA was also observed in PB-treated normal rats. These results indicate that PB and Dex have an additional effect of increasing the hepatic level of the specific mRNA of P450-male/(M-1) or a closely related form. Noncoordinate changes in the level of P450-male protein and mRNA also suggest that the hepatic level of P450-male protein is regulated by plural mechanisms: pretranslational and translational regulation in which pituitary growth hormone and/or other endocrine factors are involved.