P450 mRNA Expression Research Articles

Toxic effects of polyethylene microplastics on transcriptional changes, biochemical response, and oxidative stress in common carp (Cyprinus carpio)

Aquatic ecosystems have become a place for accumulating microplastics (MPs). MPs can directly or indirectly damage organisms. Although studies of the toxicity of MPs, there are insufficient literature reports on the effects of MPs on freshwater aquatic life. Therefore, this study aimed to evaluate the effect of MPs toxicity on Cyprinus carpio. In this study, biochemical parameters, oxidative biomarkers, and gene expression were assayed in fish exposed to 0, 175, 350, 700, and 1400 μg L−1 of MPs for 30 days. MPs were detected in the liver and intestine of fish using FTIR-analysis. Mt1, Ces2, and P450 mRNA expression were enhanced in the hepatocytes of fish exposed to MPs, while Mt2 gene expression was significantly decreased. After exposure to MPs, MDA and carbonyl protein levels were higher than those of the reference group. The antioxidant capacity and glycogen contents in the hepatocytes significantly declined. MPs significantly inhibited glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G6PDH), and catalase (CAT) activities. However, superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities increased. MPs decreased the total protein, globulin levels, and butyrylcholinesterase (BChE) activity in blood. In contrast, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and creatine phosphokinase (CPK) activities increased in treated-fish with MPs. Glucose, creatinine, cholesterol and triglyceride concentrations in fish exposed to MPs were significantly higher than that of the reference group. Consequently, MPs exposure could disrupt biochemical homeostasis, oxidative stress and alter the expression of genes involved in detoxification.

Long-term oral administration of Paederia foetida decreases cytochrome P450 mRNA expression: The predictive approaches in a rat model

Long-term oral administration of Paederia foetida decreases cytochrome P450 mRNA expression: The predictive approaches in a rat model

Salinomycin induces cell cycle arrest and apoptosis and modulates hepatic cytochrome P450 mRNA expression in HepG2/C3a cells

Salinomycin (SAL) is a monocarboxylic polyether ionophore antibiotic isolated from Streptomyces albus. It exhibits an effective antitumor potential against numerous human cancer cells. This study aimed to assess the antiproliferative effects of SAL in human hepatocellular carcinoma HepG2/C3a cell line. We investigated the effects of SAL on cell growth, DNA damage induction, cell cycle changes and apoptosis; and relative changes in expression of cell cycle-related, apoptosis-related, and CYP450 genes. SAL induced cell cycle arrest in the G2/M phase, upregulation of CDKN1A and GADD45A and downregulation of cyclin genes including CCNB1 and CCNA2. SAL effectively suppressed mRNA levels of CTNNB1 gene, an important oncogene that promotes tumorigenesis. The decrease of HepG2/C3A cells’ survival can also be due to downregulation of antiapoptotic BCL-2 expression, thus promoting the induction of apoptosis by SAL. This study also demonstrated the ability of SAL in modulating hepatic cytochrome P450 (CYP) mRNA expression, such that SAL caused the upregulation of CYP1A members and CYP3A5; and downregulation of CYP3A4. Taken together, these data contribute to the understanding of the mechanism of action of SAL, highlighting that metabolizing enzymes modulated by SAL can interfere with chemotherapy treatment and it must be considered in associated treatments.

Interleukin-1β and tumor necrosis factor-α affect cytochrome P450 expression in cynomolgus macaque hepatocytes

The cynomolgus macaque, partly due to its evolutionary closeness to humans, is an important nonhuman primate species used in drug metabolism studies. In humans, expressions of cytochromes P450 (P450s), including the important drug-metabolizing enzyme P450 3A4, are affected by various cytokines. However, this phenomenon has not been fully investigated in cynomolgus macaques. In this study, the effects of cytokines on P450 expression were investigated using the quantitative polymerase chain reaction to evaluate mRNA expression. Hepatocytes from cynomolgus macaques were treated with lipopolysaccharide and various cytokines, including interleukin (IL)-1β, IL-2, IL-6, interferon-γ, and tumor necrosis factor-α, and the expression levels of 11 P450s were compared with those of solvent-treated controls. Tumor necrosis factor-α significantly decreased cynomolgus P450 2C8 and 2C76 mRNA expression in multiple lots of cynomolgus hepatocytes investigated. IL-1β significantly decreased cynomolgus P450 1A1, 2C8, 2C19, and 2C76 mRNA expression, but increased P450 3A5 mRNA expression in multiple lots of hepatocytes. Moreover, P450 1A1-and 2C19-mediated drug oxidations were significantly and dose-dependently suppressed by IL-1β, under the present limited conditions. These results suggest that cytokines can influence hepatic P450 mRNA expression levels in cynomolgus macaques, just as cytokines are reported to affect P450 expression in humans.

Alteration of avian hepatic cytochrome P450 gene expression and activity by certain feed additives.

We investigated the effect of four feed additives, namely β-glucan, a drinking water acidifier (DWA), a sanguinarine-containing product (SN) and fulvic acid, on hepatic cytochrome P450 (CYP) mRNA expression and CYP enzyme activity in chickens. The test substances were given to the chickens in the recommended dose or in tenfold dose. The administration of 5 mg/kg body weight (bw) β-glucan and 0.1 ml/kg bw DWA for five days decreased the relative gene expression of CYP1A4 and CYP2C23a. The dosing of 50 mg/kg bw β-glucan, 5 and 50 mg/kg bw SN, 1 ml/kg bw DWA and 250 mg/kg bw fulvic acid doubled the hepatic CYP1A4 activity. The activity of CYP2C and CYP3A remained unchanged. Avoidance of CYP1A-mediated feed-drug interactions requires accurate dosing of β-glucan, DWA and fulvic acid. According to our results, no treatment resulted in excessive or less CYP2C and CYP3A protein formation, which reduces the risk of potential feed additive-drug interactions in chickens. However, the administration of feed additive SN containing a plant alkaloid should be avoided concomitantly with CYP1A-metabolised medicines.

Chronic intake of high-dose of blueberry leaf extract does not augment the harmful effects of ethanol in rats.

Excessive alcohol consumption is a risk factor for liver diseases. Enhancement of alcohol metabolism could be an effective strategy to prevent these adverse effects since it promotes the clearance of ethanol and acetaldehyde from the serum. Polyphenol-rich products have shown to protect against alcohol-related liver damage. Blueberry leaves have attracted attention as they are rich polyphenols such as proantocyanidins and chlorogenic acid. In this study, we investigated the effects of a high dose of blueberry leaf extract (BLEx) on alcohol metabolism during chronic intake of ethanol. Seven-week old Sprague-Dawley (SD) rats were divided into four groups: normal liquid diet group (NLD), normal liquid diet + BLEx group (NLD + BLEx), alcohol liquid diet group (ALD), and alcohol liquid diet + BLEx (ALD + BLEx). Then, rats were fed experimental diet for 5 weeks and at the end of feeding period, body weight, food intake, liver weight, indices of liver injury, expression and activity of alcohol metabolism-related and anti-oxidative enzymes, and levels of carbonyl protein, triglyceride (TG), and total cholesterol (T-Chol) were measured. Body weight and food intake decreased, whereas liver aldehyde dehydrogenase (ALDH) activity, liver microsomal cytochrome P450 2E1 (CYP2E1) protein and mRNA expression, and heme oxygenase 1 (HO-1) mRNA expression were upregulated by ethanol intake. Dietary BLEx, however, did not affect any of these ethanol-related changes. Indices of liver injury, expression and activity of other alcohol metabolism-related enzymes, liver carbonyl protein, TG, and T-Chol levels were not altered by ethanol and BLEx. Thus, chronic BLEx intake does not ameliorate the harmful effects of ethanol.

In vitro effects of rebaudioside A, stevioside and steviol on porcine cytochrome p450 expression and activity

The physiological effects of the Stevia-derived compounds, rebaudioside A, stevioside and steviol have been the focus of several studies due to their use as sweeteners in food. Despite that, little is known about their potential food-drug interactions. In the present study, IPEC-J2 cells and primary hepatocytes were used to investigate the effect of rebaudioside A, stevioside and steviol on cytochrome p450 (CYP) mRNA expression. Moreover, hepatic microsomes were used to investigate direct interactions between the compounds and specific CYP activity. In IPEC-J2 no changes in mRNA expression of CYP1A1 or CYP3A29 were observed with the Stevia-derived compounds. In primary hepatocytes all three tested compounds induced a significant increase in CYP3A29 expression. The tested compounds had no direct effect on specific CYP activity. In conclusion, rebaudioside A, stevioside and steviol induce only minor or no changes to the CYP expression and activity, and are not likely to cause food-drug interactions.

Polybrominated diphenyl ethers (PBDEs) and hydroxylated PBDE metabolites (OH-PBDEs) in maternal and fetal tissues, and associations with fetal cytochrome P450 gene expression

BackgroundHuman fetal exposures to polybrominated diphenyl ethers (PBDEs) and their metabolites (OH-PBDEs) are unique from adults, and in combination with a different metabolic profile, may make fetal development more sensitive to adverse health outcomes from these exposures. However, we lack data to characterize human fetal PBDE exposures and the metabolic factors that can influence these exposures. ObjectiveWe examined differences between 2nd trimester maternal and fetal exposures to PBDEs and OH-PBDEs. We also characterized fetal cytochrome P450 (CYP) mRNA expression and its associations with PBDE exposures. MethodsWe collected paired samples of maternal serum and fetal liver (n=86) with a subset having matched placenta (n=50). We measured PBDEs, OH-PBDEs, and mRNA expression of CYP genes (e.g. CYP1A1, -2E1, -2J2, -2C9) in all samples. As a sensitivity analysis, we measured PBDEs and OH-PBDEs in umbilical cord serum from a subset (n=22). ResultsBDE-47 was detected in ≥96% of all tissues. Unadjusted ∑PBDEs concentrations were highest in fetal liver (geometric mean (GM)=0.72ng/g), whereas lipid-adjusted concentrations were highest in cord serum (111.12ng/g lipid). In both cases, fetal concentrations were approximately two times higher than maternal serum levels (GM=0.33ng/g or 48.75ng/g lipid). ΣOH-PBDEs were highest in maternal and cord sera and 20–200 times lower than PBDE concentrations. In regression models, maternal BDE-47 explained more of the model variance of liver than of placenta BDE-47 concentrations (adjusted R2=0.79 vs 0.48, respectively). In adjusted logistic regression models, ∑PBDEs were positively associated with expression of CYP2E1 and -2J2 (placenta), and -1A1 (liver) (p<0.05). ConclusionOur findings suggest that under normal conditions of mid-gestation, the human fetus is directly exposed to concentrations of PBDEs that may be higher than previously estimated based on maternal serum and that these exposures are associated with the expression of mRNAs coding for CYP enzymes. These results will help frame and interpret findings from studies that use maternal or cord blood as proxy measures of fetal exposures, and will inform the molecular pathways by which PBDEs affect human health.

E3‐bglobin‐Dd mice: A new model to study hepatic zonal regulation of sex‐biased cytochrome p450 mRNA expression

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and is the third leading cause of cancer death overall. In addition, HCC rates are much higher in males than in females. Our lab primarily focuses on liver genetic regulation and how dysregulated genes contribute to liver development and disease, including HCC.The cytochrome p450 (Cyp) family of enzymes are expressed mainly in the liver and have a critical role in the metabolism and biotransformation of endogenous and xenobiotic compounds. Many Cyps are expressed zonally in the livers of both mice and humans and, in addition, have been found to exhibit sex‐biased expression.Our lab has developed a novel model to study zonal hepatocyte populations within the liver. We have placed the H‐2Dd reporter gene under control of pericentrally active alpha‐fetoprotein E3 enhancer. Using antibodies against the Dd protein, which is expressed on the cell surface, we can separate pericentral and periportal hepatocytes using flow cytometry. This system enables us to analyze previously identified and novel zonally regulated genes. Using this system, we have identified Cyps that are both sex‐biased and zonally restricted within the mouse liver. Interestingly, the highly related Cyp2a4 and Cyp2a5 are female‐biased genes expressed periportally and pericentrally, respectively. These two genes have 97% genomic sequence identity over a 30 Kb region, providing a model to study sex‐biased and zonal regulation in the liver. Our central hypothesis is that sex‐biased zonally regulated Cyp enzymes contribute to sex‐biased HCC development. Understanding the roles of sex‐biased pericentral and periportal genes may lead to new insights into the basis for HCC predominance in males.

Colonization by non-pathogenic bacteria alters mRNA expression of cytochromes P450 in originally germ-free mice.

Gut microbiota provides a wide range of beneficial function for the host and has an immense effect on the host's health state. It has also been shown that gut microbiome is often involved in the biotransformation of xenobiotics; however, the molecular mechanisms of the interaction between the gut bacteria and the metabolism of drugs by the host are still unclear. To investigate the effect of microbial colonization on messenger RNA (mRNA) expression of liver cytochromes P450 (CYPs), the main drug-metabolizing enzymes, we used germ-free (GF) mice, lacking the intestinal flora and mice monocolonized by non-pathogenic bacteria Lactobacillus plantarum NIZO2877 or probiotic bacteria Escherichia coli Nissle 1917 compared to specific pathogen-free (SPF) mice. Our results show that the mRNA expression of Cyp1a2 and Cyp2e1 was significantly increased, while the expression of Cyp3a11 mRNA was decreased under GF conditions compared to the SPF mice. The both bacteria L. plantarum NIZO2877 and E. coli Nissle 1917 given to the GF mice decreased the level of Cyp1a2 mRNA and normalized it to the control level. On the other hand, the colonization by these bacteria had no effect on the expression of Cyp3a11 mRNA in the liver of the GF mice (which remained decreased). Surprisingly, monocolonization with chosen bacterial strains has shown a different effect on the expression of Cyp2e1 mRNA in GF mice. Increased level of Cyp2e1 expression observed in the GF mice was found also in mice colonized by L. plantarum NIZO2877; however, the colonization with probiotic E. coli Nissle 1917 caused a decrease in Cyp2e1 expression and partially restored the SPF mice conditions.

Use of RNA-seq to determine variation in canine cytochrome P450 mRNA expression between blood, liver, lung, kidney and duodenum in healthy beagles.

RNA sequencing (RNA-seq) is a powerful tool for the evaluation and quantification of transcriptomes and expression patterns in animals, tissues, or pathological conditions. The purpose of this study was to determine the physiologic expression of cytochrome P450 (CYP) mRNA transcripts in whole blood, kidney, duodenum, liver, and lung in healthy, adult male (n=4) and female (n=4) beagles via RNA-seq. mRNA expression was above background (transcripts per million) for 45 canine CYPs, with liver, duodenum, and lung expressing a high number of xenobiotic metabolizing CYPs, while prominent endogenous metabolizing CYP expression was present in blood and kidney. The relative expression pattern of CYP2A13, 2B11, 2C21, 2D15, 2E1, 3A12, and 27A1 in liver, lung, and duodenum was verified through qPCR. This is the first global profiling of physiologic CYP mRNA expression in multiple canine tissues, providing a platform for further studies characterizing canine CYPs and changes in gene expression in disease states.

Cellular Pathophysiology of an Adrenal Adenoma-Associated Mutant of the Plasma Membrane Ca(2+)-ATPase ATP2B3.

Adrenal aldosterone-producing adenomas (APAs) are a main cause for primary aldosteronism leading to arterial hypertension. Physiologically, aldosterone production in the adrenal gland is stimulated by angiotensin II and high extracellular potassium. These stimuli lead to a depolarization of the plasma membrane and, as a consequence, an increase of intracellular Ca(2+). Mutations of the plasma membrane Ca(2+)-ATPase ATP2B3 have been found in APAs with a prevalence of 0.6%-3.1%. Here, we investigated the effects of the APA-associated ATP2B3(Leu425_Val426del) mutation in adrenocortical NCI-H295R and human embryonic kidney (HEK-293) cells. Ca(2+) measurements revealed a higher basal Ca(2+) level in cells expressing the mutant ATP2B3. This rise in intracellular Ca(2+) was even more pronounced under conditions with high extracellular Ca(2+) pointing to an increased Ca(2+) influx associated with the mutated protein. Furthermore, cells with the mutant ATP2B3 appeared to have a reduced capacity to export Ca(2+) suggesting a loss of the physiological pump function. Surprisingly, expression of the mutant ATP2B3 caused a Na(+)-dependent inward current that strongly depolarized the plasma membrane and compromised the cytosolic cation composition. In parallel to these findings, mRNA expression of the cytochrome P450, family 11, subfamily B, polypeptide 2 (aldosterone synthase) was substantially increased and aldosterone production was enhanced in cells overexpressing mutant ATP2B3. In summary, the APA-associated ATP2B3(Leu425_Val426del) mutant promotes aldosterone production by at least 2 different mechanisms: 1) a reduced Ca(2+) export due to the loss of the physiological pump function; and 2) an increased Ca(2+) influx due to opening of depolarization-activated Ca(2+) channels as well as a possible Ca(2+) leak through the mutated pump.

Radix Puerariae lobatae (Gegen) suppresses the anticoagulation effect of warfarin: a pharmacokinetic and pharmacodynamics study.

BackgroundRadix Salvia miltiorrhiza (Danshen) and Radix Puerariae lobatae (Gegen) are used in Traditional Chinese Medicine to treat cardiovascular diseases. However, adverse herb-drug interactions were observed between warfarin and herbal remedies containing Danshen and Gegen. This study aims to investigate the pharmacokinetic and pharmacodynamic interactions between warfarin and the different components found in Danshen and Gegen.MethodsSixty Sprague–Dawley rats were used to investigate the effects of warfarin (0.2 mg/kg), Danshen (240 or 480 mg/kg) and Gegen (240 or 480 mg/kg) both in isolation and combination. The rats in the warfarin and Danshen/Gegen combination groups were given an oral dose of Danshen or Gegen 2 h after being given an oral dose of warfarin. After five consecutive days of treatment, the pharmacokinetic interactions between Danshen/Gegen and warfarin were investigated by simultaneously monitoring and comparing the cytochrome P450 (CYP) activities, mRNA and protein expression levels in the livers of the rats from the different treatment groups. The pharmacodynamic interactions were evaluated by monitoring and comparing the vitamin K epoxide reductase (VKOR) activities, mRNA and protein expression levels in the livers of rats from the different groups, as well as the thrombomodulin (TM) activities, mRNA and protein in the lungs of these animals. The rat plasma soluble thrombomodulin concentrations of the different treatment groups were also evaluated. Microsomes incubation, Real Time-Polymerase Chain Reaction and Western blot was applied respectively to study the activity, mRNA expression and protein expression of CYP, VKOR and TM.ResultsThe activities and expression levels of the CYP and VKOR enzymes in the warfarin-Gegen combination groups increased by nearly 30 % (P = 0.02) compared with the warfarin-alone group, whereas those of TM decreased by almost 25 % (P = 0.02). The administration of Danshen did not lead to any changes in the activities or the expression levels of the CYP, VKOR or TM enzymes compared with those of the control group. Gegen induced several warfarin-metabolizing CYP enzymes and neutralized the effects of warfarin towards VKOR and TM.ConclusionGegen, rather than Danshen at the same tested dosage, offsets the anticoagulant effects of warfarin by accelerating the phase I liver metabolism of warfarin, as well as increasing the activity, mRNA and protein expression of VKOR while decreasing those of TM.Electronic supplementary materialThe online version of this article (doi:10.1186/s13020-016-0078-9) contains supplementary material, which is available to authorized users.

Do biomarkers of exposure and effect correlate with internal exposure to PAHs in swine?

Humans are commonly exposed to polycyclic aromatic hydrocarbons (PAHs), a family of compounds present as mixtures in the environment. This study exposed swine to PAH mixtures in single and subacute dose regimens and collected liver and ileum tissue to measure cytochrome P450 mRNA expression and enzyme activity as biomarkers of exposure and DNA adducts and oxidized proteins as biomarkers of effect. Micronucleated reticulocytes were measured as systemic biomarkers of effect. Duration of exposure did not influence biomarkers of exposure, though exposure duration produced significant increases in DNA adducts and oxidative stress. Micronucleated reticulocyte numbers were not affected by exposure length.

Carbocyclic functionalization of quinoxalines, their chalcogen congeners 2,1,3-benzothia/selenadiazoles, and related 1,2-diaminobenzenes based on nucleophilic substitution of fluorine

Previously unknown mono-, di- and in some cases tri- and tetra- carbocycle-substituted quinoxalines (2–8), 2,1,3-benzothiadiazoles (11, 12, 14–17) and 2,1,3-benzoselenadiazoles (20-25) were synthesized by nucleophilic substitution of fluorine in 5,6,7,8-tetrafluoroquinoxaline (1) and 4,5,6,7-tetrafluoro-2,1,3-benzothia/selenadiazoles (10 and 19, respectively) with methoxide and dimethylamine. In the 1:1 reactions, the nucleophiles attacked selectively the position 6 of 1 or the position 5 of 10 and 19. The regioselective nature of the 1:1 reactions was confirmed by the DFT calculations at the M06-2X/6-31+G(d,p) level of theory. Disubstituted quinoxaline (28), thia- (29) and selena- (30) diazoles bearing two different substituents, i.e. MeO- and Me2N-, were synthesized in a similar way. New substituted 1,2-diaminobenzenes (31–33) were prepared by reduction of corresponding thiadiazoles (12, 14, 15) and isolated in the form of hydrochlorides. Compound 33 was converted into a new quinoxaline (34) by reaction with (PhCO)2. Compounds 5, 7 and 14 were studied for cytotoxicity toward the human cancer cells and effects on the cytochrome P450 mRNA expression. They did not cause any significant modulations in the expression of several cytochrome P450 genes, and 7 was weakly toxic for the Hep2 (carcinoma) and U937 (leukemia) cells, particularly, apoptosis was observed.

Strain differences in cytochrome P450 mRNA and protein expression, and enzymatic activity among Sprague Dawley, Wistar, Brown Norway and Dark Agouti rats.

Rat cytochrome P450 (CYP) exhibits inter-strain differences, but their analysis has been scattered across studies under different conditions. To identify these strain differences in CYP more comprehensively, mRNA expression, protein expression and metabolic activity among Wistar (WI), Sprague Dawley (SD), Dark Agouti (DA) and Brown Norway (BN) rats were compared. The mRNA level and enzymatic activity of CYP1A1 were highest in SD rats. The rank order of Cyp3a2 mRNA expression mirrored its protein expression, i.e., DA>BN>SD>WI, and was similar to the CYP3A2-dependent warfarin metabolic activity, i.e., DA>SD>BN>WI. These results suggest that the strain differences in CYP3A2 enzymatic activity are caused by differences in mRNA expression. Cyp2b1 mRNA levels, which were higher in DA rats, did not correlate with its protein expression or enzymatic activity. This suggests that the strain differences in enzymatic activity are not related to Cyp2b1 mRNA expression. In conclusion, WI rats tended to have the lowest CYP1A1, 2B1 and 3A2 mRNA expression, protein expression and enzymatic activity among the strains. In addition, SD rats had the highest CYP1A1 mRNA expression and activity, while DA rats had higher CYP2B1 and CYP3A2 mRNA and protein expression. These inter-strain differences in CYP could influence pharmacokinetic considerations in preclinical toxicological studies.

Effect of weaning age on cortisol release in piglets

The effect of weaning age on the adrenal cortex, which plays a vital role in the stress response, is currently unknown. Therefore, plasma adrenocorticotropic hormone (ACTH) and cortisol levels, weights and relative weights of adrenal glands, and steroidogenesis-related protein and enzyme expression levels in piglets weaned on different days were determined. Piglets weaned at 35 days had significantly lower ACTH levels than those weaned at 14 or 21 days, and cortisol levels of piglets weaned at 21, 28, and 35 days were significantly lower than those of piglets weaned on day 14. Adrenal gland weights of piglets weaned at 28 and 35 days and relative adrenal gland weights of piglets weaned at 35 days were significantly lower than those of piglets weaned at 14 days. However, no significant difference was detected in the expression of melanocortin-type 2 receptor mRNA, which is associated with weaning age. Steroidogenic acute-regulatory (StAR) mRNA and cholesterol side-chain cleavage cytochrome P450 mRNA expression levels in piglets weaned at 28 and 35 days were significantly lower than in those weaned at 14 or 21 days, and P450 11β mRNA expression levels in piglets weaned at 28 and 35 days were significantly lower than in those weaned at 14 days. Therefore, early-weaned piglets exhibited increased adrenal gland weights and StAR and steroidogenic enzyme expression, all of which contributed to high cortisol levels. The high plasma ACTH and cortisol levels in early-weaned piglets indicate that these animals would be greatly affected by stress.

Estrogen synthesis and secretion during postnatal testicular development in male goats: In situ aromatase mRNA expression

The role of testosterone in males has been extensively studied. However, studies in several species suggest that estrogens are also important for the development and function of male brain, bone, gonads as well as for spermatogenesis and sexual behavior. Data related to testicular P450-aromatase expression and estrogen receptors α and β in several species had been published. However, to our knowledge there is no information on testicular mRNA aromatase expression, estrogen synthesis, or estradiol-17β concentrations in the circulation of the male goat. Thus, the objective of this work was to conduct in vivo and in vitro studies related to estrogen synthesis and serum concentrations during the postnatal testicular development in goat bucks. To do this, circulating concentrations of estradiol-17β and testosterone were evaluated in male goat during the first year of life, starting at 15 days of age. Besides, in order to assess the estrogen-producing ability of testicles from prepubertal male goats (four months), testicular cells were incubated with tritiated androgen precursors (3H-androstenedione and 3H-testosterone) in the presence or absence of the P450-aromatase inhibitor formestane, using TLC to identify sex steroids. Finally, the expression and tissue distribution of P450-aromatase mRNA in the testis of 3–5 month old males was evaluated by in situ RT-PCR. Results indicate that estradiol-17β is present in the serum of male goats at 0.5 months of age, and that there are significant increases at 3 and 10 months of age. The in vitro synthesis of tritiated estradiol-17β was inhibited by the P450-aromatase inhibitor formestane. Messenger RNA for P450-aromatase was abundantly expressed both in Sertoli cells in the testis of goats of ages three, four and five months. The expression of P450 mRNA was also localized in Leydig cells. It is concluded that the prepubertal goat has measurable quantities of serum estradiol-17β and that their testis has the capacity to synthesize estrogens during this period.

Effect of Coleus forskohlii and its major constituents on cytochrome P450 induction

Coleus forskohlii Briq. has been used traditionally for the treatment of several ailments since antiquity in Ayurveda. In the present study, an approach has been made to evaluate the effect of C. forskohlii and its major constituents on cytochrome P450 (CYP3A, CYP2B, and CYP2C) mRNA expression in rat hepatocytes. To gain better understanding of the herb–drug interaction potential of the chemical constituents present in C. forskohlii, the extract was subjected to column chromatography followed by standardization with respect to forskolin, 1-deoxyforskolin, and 1,9-dideoxyforskolin using reversed-phase high-performance liquid chromatography (RP-HPLC). Hepatocytes were treated with extracts, fractions, and phytoconstituents, followed by extraction and purification of total mRNA. Study of mRNA expression was carried out through reverse transcription polymerase chain reaction, followed by agarose gel electrophoresis. Results revealed that the test substances did not show any significant mRNA expression compared to the control against CYP3A, CYP2B, and CYP2C. Positive controls such as dexamethasone and rifampin showed significantly high (p < 0.001) induction potential compared to the control. It can be concluded that C. forskohlii and its major constituents may not be involved in CYP450 induction-based drug interaction.

Effect of erythropoietin on hepatic cytochrome P450 expression and function in an adenine-fed rat model of chronic kidney disease.

Erythropoietin (EPO) is used to treat anaemia associated with chronic kidney disease (CKD). Hypoxia is associated with anaemia and is known to cause a decrease in cytochrome P450 (P450) expression. As EPO production is regulated by hypoxia, we investigated the role of EPO on P450 expression and function. Male Wistar rats were subjected to a 0.7% adenine diet for 4 weeks to induce CKD. The diet continued for an additional 2 weeks while rats received EPO by i.p. injection every other day. Following euthanasia, hepatic P450 mRNA and protein expression were determined. Hepatic enzyme activity of selected P450s was determined and chromatin immunoprecipitation was used to characterize binding of nuclear receptors involved in the transcriptional regulation of CYP2C and CYP3A. EPO administration decreased hepatic mRNA and protein expression of CYP3A2 (P < 0.05), but not CYP2C11. Similarly, EPO administration decreased CYP3A2 protein expression by 81% (P < 0.001). A 32% decrease (P < 0.05) in hepatic CYP3A enzymatic activity (Vmax ) was observed for the formation of 6βOH-testosterone in the EPO-treated group. Decreases in RNA pol II recruitment (P < 0.01), hepatocyte nuclear factor 4α binding (P < 0.05) and pregnane X receptor binding (P < 0.01) to the promoter region of CYP3A were also observed in EPO-treated rats. Our data show that EPO decreases the expression and function of CYP3A, but not CYP2C in rat liver.