Microsomal Content Research Articles

Association of cytochrome P450 2D15 (CYP2D15) nonsynonymous polymorphisms and exon 3 deleted RNA splice variant with CYP2D15 protein content and enzyme function in dog liver microsomes.

CYP2D15 is a major drug metabolizing P450 in canine liver. Like the human orthologue (CYP2D6), this enzyme is highly polymorphic with at least five common nonsynonymous variants reported that result in amino acid changes, including p.Ile109Val, p.Leu115Phe, p.Gly186Ser, p.Ile250Phe and p.Ile307Val. Furthermore, a mRNA splice variant of CYP2D15 has been found in canine liver that lacks the exon 3 gene region resulting in an inactive enzyme. The objective of this study was to evaluate whether any of these amino acid variants or the exon 3 deletion mRNA variant (exon3-delta) was associated with differences in CYP2D15-selective activities or protein content in a bank of canine livers. Livers were obtained from 25 Beagles and 34 dogs of various other breeds. CYP2D15-selective activities measured included dextromethorphan o-demethylation and tramadol o-demethylation. Reverse transcription PCR showed that 76% of livers (44/58) expressed both exon3-delta and normally spliced CYP2D15 RNA, while the remaining 24% (14/58) expressed only normally spliced RNA. The presence of exon3-delta was not correlated with CYP2D15 activities or protein content. Compared with wild-type livers, Beagle dog livers heterozygous for the p.Ile109Val and p.Gly186Ser variants showed from 40 to 50% reductions in median enzyme activities, while heterozygous p.Gly186Ser livers were associated with a 41% reduction in median CYP2D15 protein content (p< .05; Dunn's test). In the entire liver bank, livers homozygous for p.Ile109Val were also associated with a 40% reduction in median dextromethorphan O-demethylation activities versus wild-type livers (p< .05). These results identify several nonsynonymous CYP2D15 gene variants associated with variable CYP2D15 metabolism in canine liver.

Evaluation on the Metabolic Activity of Two Carboxylesterase Isozymes in Mouse Liver Microsomes by a LC-MS/MS Method.

An applicable method for the precise measurement of major carboxylesterase (CESs) activity in liver still limited. Clopidogrel and irinotecan are specific substrates for CES1 and CES2, respectively. Clopidogrel is metabolized to the inactive metabolite clopidogrel carboxylate (CCAM) by CES1. Irinotecan is metabolized to the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) by CES2. In the present study, the LC-MS/MS method for the determination of CCAM and SN-38 were separately developed to characterize the metabolic activities of CES1 and CES2 in mouse liver microsomal. CCAM was separated on a Ecosil ODS column with an isocratic mobile phase consisted of 5mmol/L ammonium formate and 0.1% formic acid in water and acetonitrile (15:85, V:V) at a flow rate of 0.4mL/min. SN-38 was separated on a Waters symmetry C18 column with an gradient mobile phase consisted of 5mmol/L ammonium formate and 0.1% formic acid in water and acetonitrile at a flow rate of 0.3mL/min. Calibration curves were linear within the concentration range of 100-20,000ng/mL for CCAM and 1-200ng/mL for SN-38. The results of method showed excellent accuracy and precision. The recovery rate, matrix effect and stability inspection results were within the acceptance criteria. The optimized incubation conditions were as follows: protein concentration of microsomes were all 0.1mg/mL, incubation time was 60min for clopidogrel and 30min for irinotecan, respectively. This method was sensitive and applicable for the determination of the activity of CESs in the mouse liver microsomes.

Characteristics of cytochrome P450-dependent metabolism in the liver of the wild raccoon, Procyon lotor.

Wildlife is exposed to a wide range of xenobiotics in the natural environment. In order to appropriately assess xenobiotic-induced toxicity in wildlife, it is necessary to understand metabolic capacities. Carnivores, in general, have low metabolic abilities, making them vulnerable to a variety of chemicals. Raccoons (Procyon lotor) in the wild have been found to have high levels of xenobiotics. However, little is known about the metabolic capacity of the cytochrome P450 (CYP) enzymes in this species. Thus, this study used liver samples to investigate the characteristics of CYP enzymes in wild raccoons. In 22 wild raccoons, CYP concentrations in hepatic microsomes were examined. To better understand the properties of CYP-dependent metabolism, in vitro metabolic activity studies were performed using ethoxyresorufin, pentoxyresorufin and testosterone as substrates. In addition, three raccoons were fed commercial dog food in the laboratory for one week, and the effects on CYP-dependent metabolism were investigated. In comparison to other mammalian species, raccoons had very low concentrations of CYP in their livers. In an in vitro enzymatic analysis, raccoons' ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-depentylase (PROD) metabolic capacities were less than one-fifth and one-tenth of rats', respectively. These results indicate the possible high risk in raccoons if exposed to high levels of environmental xenobiotics because of their poor CYP activity. In this study, the features of CYP-dependent metabolism in wild raccoons are described for the first time.

Pharmacokinetics, metabolism and off-target effects in the rat of 8-[(1H- benzotriazol-1-yl)amino]octanoic acid, a selective inhibitor of human cytochrome P450 4Z1: β-oxidation as a potential augmenting pathway for inhibition

8‐[(1H‐1,2,3‐benzotriazol‐1‐yl)amino]octanoic acid (8-BOA) was recently identified as a selective and potent mechanism-based inactivator (MBI) of breast cancer-associated CYP4Z1 and exhibited favourable inhibitory activity in vitro, thus meriting in vivo characterization. The pharmacokinetics and metabolism of 8-BOA in rats was examined after a single IV bolus dose of 10 mg/kg. A biphasic time-concentration profile resulted in relatively low clearance and a prolonged elimination half-life. The major circulating metabolites identified in plasma were products of β-oxidation; congeners losing two and four methylene groups accounted for >50% of metabolites by peak area. The –(CH2)2 product was characterized previously as a CYP4Z1 MBI and so represents an active metabolite that may contribute to the desired pharmacological effect. Ex vivo analysis of total CYP content in rat liver and kidney microsomes showed that off-target CYP inactivation was minimal; liver microsomal probe substrate metabolism also demonstrated low off-target inactivation. Standard clinical chemistries provided no indication of acute toxicity. In silico simulations using the free concentration of 8-BOA in plasma suggested that the in vivo dose used here may effectively inactivate CYP4Z1 in a xenografted tumour.

Effects of alcohol-induced increase in CYP2E1 content in human liver microsomes on the activity and cooperativity of CYP3A4

We investigate the effect of the alcohol-induced increase in the content of CYP2E1 in human liver microsomes (HLM) on the function of CYP3A4. Membrane incorporation of the purified CYP2E1 into HLM considerably increases the rate of metabolism of 7-benzyloxyquinoline (BQ) and attenuates the homotropic cooperativity observed with this CYP3A4-specific substrate. It also eliminates the activating effect of α-naphthoflavone (ANF) seen in some HLM samples. To probe the physiological relevance of these effects, we compared three pooled preparations of HLM from normal donors (HLM-N) with a pooled preparation from ten heavy alcohol consumers (HLM-A). The composition of the P450 pool in all samples was characterized by the mass-spectrometric determination of 11 cytochrome P450 species. The fractional content of CYP2E1 in HLM-A was from 2.0 to 3.4 times higher than in HLM-N. In contrast, the content of CYP3A4 in HLM-A was the lowest among all samples. Despite that, HLM-A exhibited a much higher metabolism rate and a lower homotropic cooperativity with BQ, similar to CYP2E1-enriched HLM-N. To substantiate the involvement of interactions between CYP2E1 and CYP3A4 in these effects, we probed hetero-association of these proteins in CYP3A4-containing Supersomes™ with a technique employing CYP2E1 labeled with BODIPY-618 maleimide. These experiments evinced the interactions between the two enzymes and revealed an inhibitory effect of ANF on their association. Our results demonstrate that the functional properties of CYP3A4 are fundamentally dependent on the composition of the cytochrome P450 ensemble and suggest a possible impact of chronic alcohol exposure on the pharmacokinetics of drugs metabolized by CYP3A4.

Development of a method to determine cytochrome P450 1A2, 2C9, 2D6 and 3A4 activity sheep hepatic microsomes

IntroductionEx vivo studies of human fetal hepatic drug metabolism are uncommon as it requires access to functional liver tissue and therefore raises practical and ethical concerns. Large animal models provide an alternative opportunity to study changes in cytochrome P450 (CYP) activity in the mother and fetus during pregnancy. We aimed to develop methods to determine the activity of CYP1A2, CYP2C9, CYP2D6 and CYP3A4 in sheep hepatic microsomes. MethodsWe identified optimal conditions to determine the activity of CYP1A2 (using the probe drug phenacetin), CYP2C9 (diclofenac), CYP2D6 (dextromethorphan) and CYP3A4 (midazolam) by varying techniques for microsome extraction, probe drug concentration, incubation time and microsome concentration. The specificity of each probe drug was assessed by determining the rate of metabolism when specific CYP enzyme inhibitors were included in the reaction. ResultsThe optimum incubation time and probe drug concentration was six hours with 5 μM phenacetin (CYP1A2), four hours with 10 μM diclofenac (CYP2C9), 30 min with 1 μM of midazolam (CYP3A4) and 10 min with 1 μM dextromethorphan (CYP2D6). For both CYP2D6 and CYP3A4 reactions required 20 μg of microsomal protein, whereas for CYP1A2 and CYP2C9, reactions required 40 μg of microsomal protein. Metabolism of phenacetin, dextromethorphan and midazolam was reduced by specific enzyme inhibitors, but the specific CYP2C9 inhibitor sulfaphenazole did not substantially inhibit diclofenac metabolism. DiscussionThis study identifies the optimal conditions for determining CYP activity in maternal sheep hepatic microsomes. In doing so, we have developed a standardised protocol for assessment of microsomal activity of CYP3A4, CYP1A2 and CYP2D6, but we were unable to optimise conditions for assessment of CYP2C9. This approach can be applied to investigate the impact of pregnancy complications on maternal and fetal hepatic drug metabolism.

Evaluation of a Competitive Equilibrium Dialysis Approach for Assessing the Impact of Protein Binding on Clearance Predictions

Fraction unbound (fu) is an important consideration when characterizing the ADME properties of drug candidates. For highly bound compounds, there can be low confidence in quantifying fu introducing uncertainty in certain parameter estimations. Specifically, predictions of clearance (CL) rely on accurate fu values measured in plasma (fu,p) and microsomes (fu,mic) to scale in vitro intrinsic CL to in vivo CL. However, determining the ratio of fu,p/fu,mic may circumvent the need to measure discrete binding values. The purpose of this study was to evaluate a plasma-to-microsome competitive equilibrium dialysis (cED) method to determine fu,p/fu,mic ratio (fuR) for nine physiochemically-distinct compounds, and to investigate the impact of altering microsomal concentrations on fuR. The values of fuR were comparable to ratios calculated from discretely measured fu,p and fu,mic values. Furthermore, increasing microsomal concentrations increased fuR for basic and neutral compounds. When using fuR values, there was a good in vitro-in vivo correlation (IVIVC) (≤3-fold observed in vivo CL). These results suggest that the cED method used to determine fuR may be an appropriate, alternative IVIVC approach. Application of cED may extend beyond IVIVC of CL to evaluate other parameters such as species differences in protein binding and free tissue to plasma ratios.

Fluorescence versus radioactivity for assaying antifungal compound inhibited yeast 1,3-β-glucan synthase activity

Yeast membrane (1,3)-β-glucan synthase (GS; EC 2.4.1.34) activity was assayed by a fluorescent dye-binding method and a conventional radioactivity method to compare its in vitro inhibition by the echinocandin-class antifungal compound. In the fluorescence method, GS-containing microsomal plasma membrane was first reacted with UDP-glucose substrate to form the 1,3-β-glucan product. The product was then solubilized and bound specifically with aniline blue to form a 1,3-β-glucan-fluorochrome dye complex for reporting the GS activity. The effect of each component in the GS assay buffer on fluorescence was examined. A calibration curve was constructed using yeast glucan as standard. In addition, the GS activities using the same amount of 1 μg/μL microsomal concentration in the two assays were compared. The radioactivity assay showed 38.3 μM UDP-glucose incorporation and corresponded to the formation of equivalent 262 μM yeast glucan in the fluorescent assay. It indicated a possible underestimation of 1,3-β-glucan products in the radioactivity assay or an overestimation of the products in the fluorescence assay. However, pneumocandin A0 exhibited an IC50 value of 1.25 μM in the fluorescent assay closely comparable with that of 1 μM in the radioactivity assay. In summary, both the fluorescence and radioactivity tests generated similar data and pneumocandin inhibited GS. Possible limitations in the radioactivity assay were also discussed.

Strategies in herbivory by mammals revisited: The role of liver metabolism in a juniper specialist (Neotoma stephensi) and a generalist (Neotoma albigula).

Although herbivory is widespread among mammals, few species have adopted a strategy of dietary specialization. Feeding on a single plant species often exposes herbivores to high doses of plant secondary metabolites (PSMs), which may exceed the animal's detoxification capacities. Theory predicts that specialists will have unique detoxification mechanisms to process high levels of dietary toxins. To evaluate this hypothesis, we compared liver microsomal metabolism of a juniper specialist, Neotoma stephensi (diet >85% juniper), to a generalist, N.albigula (diet ≤30% juniper). Specifically, we quantified the concentration of a key detoxification enzyme, cytochrome P450 2B (CYP2B) in liver microsomes, and the metabolism of α-pinene, the most abundant terpene in the juniper species consumed by the specialist woodrat. In both species, a 30% juniper diet increased the total CYP2B concentration (2-3×) in microsomes and microsomal α-pinene metabolism rates (4-fold). In N.stephensi, higher levels of dietary juniper (60% and 100%) further induced CYP2B and increased metabolism rates of α-pinene. Although no species-specific differences in metabolism rates were observed at 30% dietary juniper, total microsomal CYP2B concentration was 1.7× higher in N.stephensi than in N.albigula (p<.01), suggesting N.stephensi produces one or more variant of CYP2B that is less efficient at processing α-pinene. In N.stephensi, the rates of α-pinene metabolism increased with dietary juniper and were positively correlated with CYP2B concentration. The ability of N.stephensi to elevate CYP2B concentration and rate of α-pinene metabolism with increasing levels of juniper in the diet may facilitate juniper specialization in this species.

Characterization of an Acyl‐CoA:retinol Acyltransferase (ARAT) Activity in the Hepatopancreas of the American Lobster (Homarus americanus)

Vitamin A (retinol) is a necessary nutrient in higher animals. In mammals, the pathways by which retinol is transported and metabolized are well described. One important reaction in these pathways is the conversion of retinol into retinyl esters, which occurs in several tissues, including the small intestine, liver, and eye. Little is known about the conversion of retinol into retinyl esters in invertebrates. We have identified an acyl‐CoA:retinol acyltransferase (ARAT) activity in the hepatopancreas of the American lobster (Homarus americanus). This activity shows a linear dependence on time and microsome concentration. It shows temperature dependence and an optimal pH of 6.5. We have characterized its acyl‐CoA preference and determined Km and Vmax values for both retinol and acyl‐CoA.Support or Funding InformationCentre College

UPLC‐MS/MS Analysis of Ethoxyresorufin O‐Deethylation (EROD) Kinetics in Rat Kidney Microsomes

PurposeFormation of resorufin from ethoxyresorufin has been widely used to assess cytochrome P450 1A1 (CYP1A1) activity. Additionally, the kinetics of CYP1A1 activity has been well characterized in the liver microsomes by spectrophotometric/chromatographic methods. However, studies in kidney microsomes are scarce, most likely due to the lower microsomal content of CYP1A1 and lower production of the resorufin in the kidneys relative to those in the liver. In the present study, we aimed to develop a microscale enzymatic incubation method, coupled with a sensitive UPLC‐MS/MS assay for the characterization of kinetic parameters of 7‐ethoxyresorufin O‐deethylation (EROD) in the kidney microsomes.MethodsKidney microsomes were prepared from adult male Sprague‐Dawley rats (n = 3) by differential centrifugation. Protein (0.1, 0.25, 0.5 mg/mL) and time (2.5, 5.0, 7.5, and 10 min) linearity experiments with regard to the rate of production of resorufin from the substrate were determined (n = 3) using a substrate concentration of 0.5 μM. For determination of the Michaelis‐Menten (MM) kinetic parameters of EROD, the reaction mixtures (50 μL), containing microsomal proteins (0.25 mg/mL) were incubated with different concentrations of ethoxyresorufin (0.1–5.0 μM) in the presence of NADPH (1 mM) for 10 min at 37°C. The reaction was terminated by the addition of 100 μL of acetonitrile, containing internal standard (d6‐resorufin). After centrifugation, the supernatant (5 μL) was injected onto a UPLC column (Phenomenex C18), and sample components were separated using a binary gradient mobile phase (A: 5 mM ammonium acetate in water and B: acetonitrile/methanol, 95:5) at a flow rate of 0.3 mL/min. The UPLC–MS/MS system used the electrospray ionization technique in positive ion mode, and the transitions of m/z 213.9→185.9 and 220→192 were used to quantitate resorufin and the IS, respectively. Analytical method validation was performed as per regulatory guidelines.ResultsThe analytical method met the acceptance criteria for method validation in the linear range of 0.5 nM to 75 nM resorufin, with a lower limit of quantitation of 0.5 nM. The intra‐ and inter‐day precision was in the range of 4.55–12.4% and 6.17–9.82%, respectively. The overall recoveries of resorufin (90%–99%) and IS (85%–103%) were relatively high, suggesting an almost complete recovery from the protein precipitation method and confirming the lack of matrix effect on the samples. Using the optimized incubation and quantitation methods, we were able to reduce the incubation volume (50 μL), microsomal protein amount (12.5 μg), and incubation time (10 min), compared with reported methods. The method was successfully applied to the estimation of the MM kinetic parameters of 7‐ethoxyresorufin O‐deethylase activity in the rat kidney microsomes (Fig 1), which showed a maximum velocity of 1.92 ± 0.25 pmol/min/mg and a MM constant of 1.24 ± 0.2 μM (mean ± SD).ConclusionThe use of a sensitive and validated UPLC‐MS/MS method allows determination of the MM kinetic parameters of CYP1A1 activity in the rat kidney microsomes.Support or Funding InformationFunding: Chapman University School of PharmacyFormation rate of resorufin from ethoxyresorufin in rat kidney microsomal incubations (n = 3)Figure 1

Network Pharmacology-Based Prediction and Verification of Qingluo Tongbi Formula to Reduce Liver Toxicity of Tripterygium wilfordii via UGT2B7 in Endoplasmic Reticulum.

BackgroundThe hepatotoxicity of Tripterygium wilfordii Hook. f. (TWHF) limits its clinic utilization. Qingluo Tongbi formula (QTF) was formulated based on a basic Chinese medicine theory. Previous studies have confirmed the safety and efficacy of QTF in treating rheumatoid arthritis. Therefore, we considered that TWHF could be detoxified based on its reasonable compatibility with QTF. We investigated the detoxicity mechanism of QTF in reducing the liver toxicity of TWHF.Material/MethodsWe used network pharmacology to determine the relevant metabolism targets of TWHF, focusing on the phase II metabolic enzymes uridine diphosphate-glucuronosyltransferase 1A1 (UGT1A1), UGT1A6, and UGT2B7. Based on the molecular mechanisms of these predictions and the results of the network analysis, we designed experiments to verify our hypothesis in vivo. We used western blotting, real-time quantitative polymerase chain reaction (RT-qPCR), double immunofluorescence, and laser confocal microscopy to detect the expression of UGTs. Finally, we used transmission electron microscopy to observe the endoplasmic reticulum structure.ResultsThe results confirmed that QTF reversed the TWHF-induced reduction of UGT content in liver microsomes, upregulated UGT1A1 and UGT1A6 but not UGT2B7 in the liver tissue. UGT2B7 expression in the liver and liver microsomes was inconsistent. QTF upregulated the expression of UGT2B7 in the endoplasmic reticulum, and QTF upregulated UGT2B7 expression levels in the endoplasmic reticulum compared with TWHF, which reduced liver toxicity. Structural changes were observed in the endoplasmic reticulum.ConclusionsThe Chinese traditional medicine compound QTF can achieve the effect of detoxification by upregulating the expression of UGT2B7 in the endoplasmic reticulum.

Pharmacokinetics of active ingredients of Pudilan Xiaoyan Oral Liquid in rats and effect on liver drug-metabolizing enzymes

This study is aimed to establish a method for the determination of baicalin,baicalin and purpurin in the plasma of rats after oral administration of Pudilan Xiaoyan Oral Liquid( PDL) by using liquid chromatography-mass spectrometry( LC-MS),analyze the pharmacokinetics of three components in rats,and investigate the effects of PDL on drug-metabolizing enzymes in rat liver. C18 column was used for liquid chromatography separation,with acetonitrile-water( containing 0. 2% formic acid) as the mobile phase for gradient elution. The mass spectrometry was detected by electrospray ion source( ESI) under multi-reaction monitoring mode( MRM),as well as positive and negative ion alternating mode. Plasma sample collection was performed by using an automatic blood collection meter for small animals. The pharmacokinetic parameters were calculated by Win Nonlin software. The total protein concentration of rat liver microsomes and the total enzyme content of CYP450 were determined by BCA method and spectrophotometry respectively. The methodological study in terms of linear range,recovery rate,precision and sample stability,was used to confirm that the LC-MS analysis method established in this experiment was simple,exclusive,accurate and reliable,and can meet the requirement of determining the content of baicalin,oroxindin and corynoline in plasma after PDL administration in rats. The drug-time curve showed that baicalin and oroxindin had a bimodal phenomenon,and the pharmacokinetic parameters indicated that baicalin,oroxindin and corynoline in PDL had certain drug-like properties. After 7 consecutive days of PDL administration,the rat liver coefficient,total liver microparticle protein and CYP450 enzyme content were increased,but there was no significant difference,indicating that PDL was less likely to develop drug-drug interaction based on CYP enzyme. The results of this experiment can provide reference for the research on in vivo efficacy and drug interaction of PDL as well as on its clinical application.

Maternal recognition of pregnancy

Enhanced secretion of PGF2 alpha from endometrial explants in vitro in response to oxytocin is associated with augmented activities of phospholipase A2, phospholipase C and prostaglandin endoperoxide H synthase (PGS). In early pregnancy, maintenance of the corpus luteum is associated with an absence of pulsatile PGF2 alpha secretion; an increase in endometrial inhibitors of phospholipase A2 and PGS contribute to the antiluteolytic alterations of PGF2 alpha secretion. Linoleic acid is a competitive inhibitor of arachidonic acid metabolism by PGS, and microsomal concentrations of free linoleic acid are increased in the endometrium of pregnant cattle. The trophoblast produces large quantities of interferon tau (IFN-tau). Inhibition of increases in endometrial oestradiol receptor mRNA and protein are associated with intrauterine administration of recombinant (r) ovine (o) IFN-tau in sheep. Intrauterine injections of ovine (b) IFN-tau in cattle (days 14-17) altered endometrial function so that secretion of PGF2 alpha from cultured endometrial epithelial cells was reduced. Antiluteolytic effects were not expressed in 20% of cows receiving IFN-tau or rbIFN-alpha I1 indicating that an inadequate endometrial responsiveness may contribute to embryo mortality. IFN-tau may activate a signal transduction system similar to that induced by other type I IFNs; activation of an intracellular tyrosine kinase ultimately leads to activation of an IFN-stimulated response element to induce gene transcription. Biological responses associated with pregnancy and IFN-tau treatment are integrated into a multifactorial antiluteolytic model. Strategies to enhance embryo survival could include supplementation with rIFN-tau and alterations in endometrial responsiveness to this cytokine through dietary manipulation of lipid metabolism.

The Impact of Scaling Factor Variability on Risk-Relevant Pharmacokinetic Outcomes in Children: A Case Study Using Bromodichloromethane (BDCM).

Biotransformation rates extrapolated from in vitro data are used increasingly in human physiologically based pharmacokinetic (PBPK) models. This practice requires use of scaling factors, including microsomal content (mg of microsomal protein/g liver, MPPGL), enzyme specific content, and liver mass as a fraction of body weight (FVL). Previous analyses indicated that scaling factor variability impacts pharmacokinetic (PK) outcomes used in adult population dose-response studies. This analysis was extended to pediatric populations because large inter-individual differences in enzyme ontogeny likely would further contribute to scaling factor variability. An adult bromodichloromethane (BDCM) model (Kenyon, E. M., Eklund, C., Leavens, T. L., and Pegram, R. A. (2016a). Development and application of a human PBPK model for bromodichloromethane (BDCM) to investigate impacts of multi-route exposure. J. Appl. Toxicol. 36, 1095-1111) was re-parameterized for neonates, infants, and toddlers. Monte Carlo analysis was used to assess the impact of pediatric scaling factor variation on model-derived PK outcomes compared with adult findings. BDCM dose metrics were estimated following a single 0.05-liter drink of water or a 20-min bath, under typical (5 µg/l) and plausible higher (20 µg/l) BDCM concentrations. MPPGL, CYP2E1, and FVL values reflected the distribution of reported pediatric population values. The impact of scaling factor variability on PK outcome variation was different for each exposure scenario, but similar for each BDCM water concentration. The higher CYP2E1 expression variability during early childhood was reflected in greater variability in predicted PK outcomes in younger age groups, particularly for the oral exposure route. Sensitivity analysis confirmed the most influential parameter for this variability was CYP2E1, particularly in neonates. These findings demonstrate the importance of age-dependent scaling factor variation used for in vitro to in vivo extrapolation of biotransformation rates.

UPLC-MS/MS analysis of dextromethorphan-O-demethylation kinetics in rat brain microsomes

Formation of dextrorphan (DXT) from dextromethorphan (DXM) has been widely used to assess cytochrome P450 2D (CYP2D) activity. Additionally, the kinetics of CYP2D activity have been well characterized in the liver microsomes. However, studies in brain microsomes are limited due to the lower microsomal content and abundance of CYP2D in the brain relative to the liver. In the present study, we developed a micro-scale enzymatic incubation method, coupled with a sensitive UPLC-MS/MS assay for the quantitation of the rate of DXT formation from DXM in brain microsomes. Rat brain microsomes were incubated with different concentrations of DXM for various times. The reaction was stopped, and the proteins were precipitated by the addition of acetonitrile, containing internal standard (d3-DXT). After centrifugation, supernatant (2 μL) was injected onto a UPLC, C18 column with gradient elution. Analytes were quantitated using triple-quadrupole MS/MS with electrospray ionization in positive ion mode. The assay, which was validated for accuracy and precision in the linear range of 0.25 nM to 100 nM DXT, has a lower limit of quantitation of 0.125 fmol on the column. Using our optimized incubation and quantitation methods, we were able to reduce the incubation volume (25 μL), microsomal protein amount (5 μg), and incubation time (20 min), compared with reported methods. The method was successfully applied to estimation of the Michaelis-Menten (MM) kinetic parameters of dextromethorphan-O-demethylase activity in the rat brain microsomes (mean ± SD, n = 4), which showed a maximum velocity of 2.24 ± 0.42 pmol/min/mg and a MM constant of 282 ± 62 μM. It is concluded that by requiring far less biological material and time, our method represents a significant improvement over the existing techniques for investigation of CYP2D activity in rat brain microsomes.

Effects of tebuconazole on cytochrome P450 enzymes, oxidative stress, and endocrine disruption in male rats.

The major objective of the present study was to determine the ability of a triazole fungicide tebuconazole to induce cytochrome P450-dependent monooxygenases, oxidative stress, and endocrine-disrupting activity using male rats treated with tebuconazole at 10, 25, and 50 mg/kg p.o. once daily for 28 days. In liver, tebuconazole dose-dependently increased microsomal contents of cytochrome P450 and cytochrome b5 and the activities of NADPH-cytochrome P450 reductase, 7-ethoxyresorufin O-deethylase, methoxyresorufin O-demethylase, pentoxyresorufin O-dealkylase, 7-ethoxycoumarin O-deethylase, aniline hydroxylase, and erythromycin N-demethylase. In kidney, tebuconazole increased 7-ethoxycoumarin O-deethylase activity without affecting other monooxygenase activities. In marked contrast to liver and kidney, tebuconazole decreased testicular 7-ethoxyresorufin O-deethylase, methoxyresorufin O-demethylase, 7-ethoxycoumarin O-deethylase, aniline hydroxylase, and erythromycin N-demethylase activities. The results of immunoblot analysis of liver microsomes of controls and tebuconazole-treated rats revealed that tebuconazole induced CYP1A1/2, CYP2B1/2, CYP2E1, and CYP3A proteins in liver. Additions of tebuconazole to liver microsomes inhibited microsomal 7-ethoxycoumarin O-deethylase activity in vitro (IC50 = 1.50-1.69 µM). Treatment of rats with tebuconazole decreased glutathione content and increased glutathione S-transferase, superoxide dismutase, catalase, and glutathione peroxidase activities in liver; increased superoxide dismutase activities in kidney and testis; but decreased glutathione S-transferase activity in testis. Treatments with tebuconazole decreased serum testosterone concentration and cauda epididymal sperm count. The present study demonstrates that tebuconazole induces a multiplicity of CYPs and oxidative stress in liver; inhibits testicular P450 and glutathione S-transferase activities; and produces anti-androgenic effects in male rats.

Effects of enrofloxacin on antioxidant system, microsomal enzymatic activity, and proteomics in porcine liver.

Enrofloxacin (EF) is a widely used fluoroquinolone, usually regarded as a safe and effective treatment for bacterial infections. Adverse effects of EF have previously been demonstrated in some species, but so far there have been no studies looking specifically at the impact of EF on pigs. In this study, three different doses of EF (5, 25 and 125mgkgbw-1 ) were administrated to Bama pigs. The results showed that lipid peroxidation of pig liver tissue occurred with all EF doses. The 125mgkg dose of EF induced catalase (CAT) and glutathione peroxidase (GSH-px) and increased CYP450 content in pig liver microsomes. The activity of microsomal NADPH-cytochrome C reductase (NCCR) was elevated at both the 25 and 125mgkg doses of EF. Microsomal erythromycin N-demethylase (ERND) and aminopyrin N-demethylase (AND) were inhibited by high doses of EF, while aniline-4-hydroxylase (AH) was unaffected. None of the EF treatments affected superoxide dismutase (SOD) or cytochrome b5 content. Antioxidases and microsomal enzymes may work together to resist the adverse effects of EF. Proteomic analysis revealed increased protein expression of carboxylesterase (CES) and alpha-enolase (ENO1) in microsomes as a stress response to EF. These results provide new information about the adverse effect of fluoroquinolones and help guide their usage more effectively in the clinic or animal breeding.

The effects of aging on hepatic microsomal scaling factor and hepatocellularity number in the horse

1. Scaling factor values for the in vitro-in vivo extrapolation of hepatic metabolic clearance for xenobiotics have not yet been determined in horses. Scaling factors were determined by comparing the total protein and or cytochrome (CYP) P450 content in microsomes and cryopreserved hepatocytes against the content in the liver.2. Microsomal protein per gram of liver (MPPGL) and hepatocellularity number per gram of liver (HPGL) using CYP P450 content method ranged 41–73 mg/gram of liver (mean= 57 mg/gram of liver, n = 39) and 146–320 × 106 cells/g of liver (mean = 227× 106 cells/g of liver, n = 18), respectively and 156–352 × 106 cells/g of liver (mean = 232× 106 cells/g of liver) using total protein method.3. A non-monotonic and inverse relationship between age and MPPGL and HPGL, respectively, was observed. Between one and 20 y of age, the liver cell size decreases as age increases. Subsequently, the cell size increases until the hepatocytes of the oldest horses approached the size found in the youngest horses. Hepatocyte density was inversely related to the size of the hepatocytes.4. This study provides the first extensive and comprehensive data demonstrating the relationship between the size of hepatocytes and HPGL in any species.

Lycopene relieves AFB1-induced liver injury through enhancing hepatic antioxidation and detoxification potential with Nrf2 activation

To evaluate the chemoprotective effect and mechanism of lycopene (LYC) on aflatoxin B1 (AFB1)-induced liver injury, forty-eight male mice were randomly allocated and treated with LYC (5 mg/kg) and/or AFB1 (0.75 mg/kg) by intragastric administration for 30 days. We found that LYC reduced AFB1-DNA adducts accumulation in liver and alleviated AFB1-induced liver lesions through suppressing AFB1-induced oxidative stress and enhancing glutathione transferase (GST)-mediated detoxification. Furthermore, Nrf2 expressions and nuclear translocation, and mRNA expressions of Nrf2 downstream target genes, including GST isoenzymes, GSH synthesis related enzymes and antioxidases were elevated by pretreatment with LYC in AFB1 intoxicated mice, demonstrating the hepatoprotective of LYC against AFB1 is likely attributed to Nrf2 signaling activation. However, LYC failed to restore the reduction of total cytochrome P450 content in hepatic microsomes and mRNA expressions of P450 1A2 and 3A4, indicating LYC has no significant effect on P450-mediated bioactivation of AFB1.