CYP1A1 mRNA Research Articles

Polydatin reduces aflatoxin-B1 induced oxidative stress, DNA damage, and inflammatory cytokine levels in mice.

This study showed the protective effect of polydatin (PD), which has an antioxidant activity against oxidative stress in mice caused by aflatoxin B1 (AFB1). In this study, 36 male Swiss albino mice were divided equally into 6 groups: 0.2mL of FTS was administered to the control group, 0.2mL of olive oil to the second group, and 0.75mg/kg AFB1 to the third group by intragastric gavage every day for 28days. The fourth, fifth, and sixth groups were administered 50, 100, and 200mg/kg PD and 0.75mg/kg AFB1 intragastrically for 28days, respectively. AFB1 administration increased plasma aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, blood urea nitrogen, creatinine, and malondialdehyde levels in blood and tissue samples but decreased the level of glutathione and the activities of superoxide dismutase and catalase. On the other hand, it was determined that PD applications depending on the increasing doses brought these levels closer to normal. In addition, AFB1 administration increased the amount of ssDNA and liver COX-2, TNF-α, IL-6, NFκB, and Cyp3a11 mRNA expression levels; on the other hand, it decreased the IL-2 mRNA expression level. In contrast, increasing doses of PD application regulated the amount of ssDNA and these mRNA expression levels. Additionally, histopathological damage was observed in the liver and kidney tissues of the AFB1 group, while PD applications in a dose-dependent manner improved these damages. As a result, it was determined that PD reduced AFB1-induced oxidative stress, DNA damage, and inflammation and exhibited a protective effect on tissues in mice.

Impact of tectoridin on the pharmacokinetics of florfenicol via targeting cytochrome P450 and P-glycoprotein of rats

Belamcanda chinensis (L.) DC, commonly used with florfenicol in Chinese veterinary clinics for respiratory tract infections, contains the major effective isoflavone, tectoridin (TEC). This study aimed to investigate the impact of TEC co-administration on the pharmacokinetics of florfenicol in vivo. Male rats received oral TEC (50 mg/kg BW) or sterile water for seven days, followed by a single oral dose of florfenicol (25 mg/kg BW) on the 8th day. Non-compartmental methods analysed the pharmacokinetics of florfenicol, while real-time reverse transcription polymerase chain reaction (RT-PCR), Western blot, and immunohistochemical analyses measured expression levels of cytochrome P450 (CYP) isoforms in the liver and P-glycoprotein (P-gp) in the jejunum. TEC significantly decreased florfenicol’s AUC(0–∞), MRT(0–∞), t 1/2z, Vz/F, and C max by 24.75%, 18.43%, 55.47%, 43.05%, and 19.48%, while increasing CLz/F by 33.33%. TEC also up-regulated hepatic CYP1A2 and CYP3A1 mRNA expression, as well as intestinal MDR1, by 1.39-fold, 1.85-fold, and 1.65-fold. This coincided with a respective increase in protein expression by 1.37-fold, 1.39-fold, and 1.43-fold. These findings suggest that TEC-induced alterations in the pharmacokinetics of florfenicol may be attributed to increased CYP and P-gp expression. Further investigations are warranted to understand the implications of these findings on the clinical effectiveness of florfenicol in veterinary practice.

Drug-drug interaction potential of SH-1028, a third-generation EGFR-TKI: in vitro and clinical trials.

SH-1028 is an irreversible third-generation EGFR tyrosine kinase inhibitor (EGFR-TKI) for the treatment of locally advanced or metastatic non-small cell lung cancer (NSCLC). Considering the possibility of combination therapy in patients with NSCLC, we investigated the drug-drug interaction (DDI) potential of SH-1028 both in vitro and in clinical trials. The in vitro studies were conducted to determine the potential of SH-1028 as a substrate, inducer, or inhibitor of cytochrome P450 (CYP) subtypes. A phase I drug-drug interaction study in healthy volunteers was performed to evaluate the impact of co-administering rifampicin (a strong CYP3A4 inducer) and itraconazole (a strong CYP3A4 inhibitor) on the pharmacokinetics of SH-1028. The in vitro experiments showed that SH-1028 was mainly metabolized by CYP3A4. The activities of CYP1A2, 2B6, 2C19, 2D6 and 3A4 enzymes were slightly inhibited in vitro with SH-1028. SH-1028 has no obvious induction effect on CYP1A2 and CYP2B6 activities, but has potential induction effect on CYP3A4 mRNA expression. However, SH-1028 may not induce or inhibit human CYPs significantly at the clinically expected dose (200mg). The geometric mean ratios of pharmacokinetic parameters and their corresponding 90% confidence intervals for SH-1028 in combination and alone did not fall within the range of 80-125%. It is speculated that itraconazole and rifampicin affect the metabolism of SH-1028. In the clinical application of SH-1028, special attention should be paid to the interaction between SH-1028 and drugs or foods that affect the activity of CYP3A4. (Clinical trial registration number: CTR20210558).

Lactobacillus fermentum MCC2760 abrogate high-fat induced perturbations in the enterohepatic circulation of bile acids in rats

This study in hyperlipidemic rats elucidated the effect of Lactobacillus fermentum MCC2760 on intestinal bile acid (BA) uptake, hepatic BA synthesis, and enterohepatic BA transporters. Diets rich in saturated fatty acids [coconut oil (CO)] and omega-6 fatty acids [sunflower oil (SFO)] at 25g fat/100g diet were fed to rats with or without MCC2760 (109 cells/kg body weight). After 60days of feeding, intestinal BA uptake and expression of Asbt, Osta/b mRNA and protein, and hepatic expression of Ntcp, Bsep, Cyp7a1, Fxr, Shp, Lrh-1, and Hnf4a mRNA were measured. Hepatic expression of HMG-CoA reductase protein and its activity and total BAs in serum, liver, and feces were assessed. Hyperlipidaemic groups (HF-CO and HF-SFO) had: 1) increased intestinal BA uptake, Asbt and Osta/b mRNA expression, and ASBT staining 2) increased BA in serum, 3) decreased hepatic expression of Ntcp, Bsep, and Cyp7a1 mRNA, and NTCP staining 4) increased activity of HMG-CoA reductase, 5) increased hepatic expression of Fxr and Shp mRNA, 6) decreased hepatic expression of Lrh-1 and Hnf4a mRNA, and 7) decreased BA in Feces when compared to their respective controls (N-CO and N-SFO) and experimental groups (HF-CO+LF and HF-SFO+LF). Immunostaining revealed increased intestinal Asbt and hepatic Ntcp protein expression in the HF-CO and HF-SFO groups compared to control and experimental groups. Incorporating probiotics like MCC2760 abrogated hyperlipidemia-induced changes in the intestinal uptake, hepatic synthesis, and enterohepatic transporters of BA in rats. Probiotic MCC2760 can be used to modulate lipid metabolism in high-fat-induced hyperlipidemic conditions.

Dicloxacillin-Warfarin Drug-Drug Interaction - A Register-Based Study and In Vitro Investigations in 3D Spheroid Primary Human Hepatocytes.

Dicloxacillin is used to treat staphylococcal infections and we have previously shown that dicloxacillin is an inducer of cytochrome P450 enzymes (CYPs). Here, we employed a translational approach to investigate the effect of a treatment with dicloxacillin on warfarin efficacy in Danish registries. Furthermore, we assessed dicloxacillin as an inducer of CYPs in vitro. We conducted a register-based study and analyzed international normalized ratio (INR) levels in chronic warfarin users before and after short- and long-term use of dicloxacillin (n=1023) and flucloxacillin (n=123). Induction of CYPs were investigated in a novel liver model of 3D spheroid primary human hepatocytes (PHHs) at the level of mRNA, and protein and enzyme activity. Short- and long-term dicloxacillin treatments decreased INR levels by -0.65 (95% CI: -0.57 to -0.74) and -0.76 (95% CI: -0.50 to -1.02), respectively. More than 90% of individuals experienced subtherapeutic INR levels (below 2) after long-term dicloxacillin treatment. Flucloxacillin decreased INR levels by -0.37 (95% CI: -0.14 to -0.60). In 3D spheroid PHHs, the maximal induction of CYP3A4 mRNA, protein and enzyme activity by dicloxacillin were 4.9-, 2.9- and 2.4-fold, respectively. Dicloxacillin also induced CYP2C9 mRNA by 1.7-fold. Dicloxacillin induces CYP enzymes and reduces the clinical efficacy of warfarin in patients. This effect is substantially exacerbated during long-term treatment with dicloxacillin. The in vitro results corroborated this drug-drug interaction and correlated to the clinical findings. Caution is warranted for warfarin patients that initiate dicloxacillin or flucloxacillin, especially for a long-term treatment of endocarditis.

Molecular cloning, expression analysis and functional characterization of chicken cytochrome P450 27A1: A novel mitochondrial vitamin D3 25-hydroxylase

Vitamin D3 is hydroxylated by cytochrome P450 (CYP) before exerting biological effects. The chicken CYP involved in vitamin D3 25-hydroxylation has yet to be cloned, and little is known about its functional characteristics, tissue distribution, and cellular expression. We identified a novel, full-length CYP27A1 gene cloned from chicken hepatocyte cDNA that encodes a putative protein of 518 amino acids. Swiss modeling revealed that chicken CYP27A1 has a classic open-fold form. Multisequence homology alignment determined that CYP27A1 contains conserved motifs for substrate recognition and binding. Quantitative real-time PCR analysis in 2-mo-old Partridge Shank broilers demonstrated that CYP27A1 mRNA levels were highest in the liver, followed by the thigh muscles, the breast muscles, and kidneys. The transcripts of CYP27A1 in breast muscles were significantly higher in males than in females. A subcellular localization analysis demonstrated that CYP27A1 was mainly expressed in the mitochondria. In vitro enzyme assays suggested that recombinant CYP27A1 hydroxylates vitamin D3 at the C-25 position to form 25-hydroxyvitamin D3 (25(OH)D3). The Km and Vmax values for CYP27A1-dependent vitamin D3 25-hydroxylation were estimated to be 4.929 μM and 0.389 mol min-1 mg-1 protein, respectively. In summary, these results suggest that CYP27A1 encodes a mitochondrial CYP that plays an important physiologic role in the 25-hydroxylation of vitamin D3 in chickens, providing novel insights into vitamin D3 metabolism in this species.

Alterations in gonadotropin, prolactin, androgen and estrogen receptor and steroidogenesis-associated gene expression in gander testes in relation to the annual period

Physiological mechanisms of seasonal changes in testicular function in birds are not fully elucidated. The balance between androgens and estrogens and testis sensitivity for gonadotropin and gonadal steroids are still unclear. The aim of the study was to examine: (1) the changes in circulating and intra-testicular steroid hormone levels and their relationship; (2) the mRNA expression of testicular gonadotropin, prolactin (PRL), progesterone (P4), androgen, and estrogen receptors, and (3) key steroidogenesis processes-related genes with immunofluorescent localization of aromatase in gander testes during the annual period. Testes from ganders (n = 25) in the first reproduction season were obtained at five breeding stages, i.e., prebreeding (PrB), peak of reproduction (PR), postbreeding (PoB), nonbreeding (NB), and onset of reproduction (OR). Males were kept under breeding conditions. It was found that plasma P4 levels decreased at the PoB and NB stages, whereas intra-testicular P4 was the highest in the NB stage. Intra-testicular estradiol (E2) levels were higher at the PoB and NB stages than the other stages, whereas testosterone (T) levels showed a nearly opposite pattern. The plasma estradiol–to–testosterone ratios were higher at the PrB, PoB and NB stages compared to other stages. The transcript abundances for luteinizing hormone receptor (LHR), PRL receptor (PRLR), estrogen receptor alpha (ERα), and estrogen receptor beta (ERβ) also change in testicular tissue during the annual period. Moreover, StAR mRNA expression was upregulated at the PoB and NB stages, and CYP11A1 transcript level was the highest at the PoB stage. Stage-dependent changes in the CYP19A1 mRNA and aromatase protein levels with higher abundances of transcript at PoB and NB stages and protein at the NB stage were observed. Localization and immunofluorescent signal intensity for aromatase also differed in relation to the examined stages. It may be suggested that differential E2 levels, as well as aromatase expression and localization across annual stages are responsible for the seasonal activation/inactivation stages of testis spermatogenesis in domestic ganders. These data strongly suggest a role of aromatase in the control of gander steroidogenesis as changes in this enzyme level are associated with alternation in gonadal steroid hormones. In addition, joint action with others hormones, like PRL and LH, seems to be important in the final effect of seasonal reproduction potential.

High-fat intake alleviates lung injury induced by Paragonimus proliferus infection in rats through up-regulating CYP 4A1 expression in lung tissues

To explore the improvements of high-fat intake on lung injury induced by Paragonimus proliferus infection in rats, and to preliminarily explore the mechanisms underlying the role of cytochrome P450 4A1 (CYP 4A1) in the improve ments. SD rats were randomly assigned into three groups, including the normal control group (n = 10), the infection and normal diet group (n = 12) and the infection and high-fat diet group (n = 12). Rats in the normal control group were fed with normal diet and without any other treatments, and animals in the infection and normal diet group were subcutaneously injected with 8 excysted metacercariae of P. proliferus via the abdominal wall, followed by feeding with normal diet, while rats in the infection and high-fat diet group were subcutaneously injected with 8 excysted metacercariae of P. proliferus via the abdominal wall, followed by feeding with high-fat diet. All rats were sacrificed 28 weeks post-infection, and serum samples and lung specimens were collected. Following hematoxylin-eosin (HE) staining of rat lung specimens, the rat lung injury was observed under an optical microscope, and alveolitis was evaluated using semi-quantitative scoring. Serum interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) levels were measured using enzyme-linked immunosorbent assay (ELISA), and the cytochrome P450 4A1 (CYP 4A1) expression was quantified in rat lung specimens at transcriptional and translational levels using quantitative real-time PCR (qPCR) and Western blotting assays. Alveolar wall thickening, edema and inflammatory cell infiltration were alleviated 28 weeks post-infection with P. proliferus in rats in the infection and high-fat diet group relative to the infection and normal diet group, and no alveolar consolidation was seen in the infection and high-fat diet group. The semi-quantitative score of alveolitis was significantly higher in the infection and normal diet group [(2.200 ± 0.289) points] than in the normal control group [(0.300 ± 0.083) points] and the infection and high-fat diet group [(1.300 ± 0.475) points] (both P values < 0.05), and higher serum IL-1β [(151.586 ± 20.492)] pg/mL and TNF-α levels [(180.207 ± 23.379) pg/mL] were detected in the infection and normal diet group than in the normal control group [IL-1β: (103.226 ± 3.366) pg/mL; TNF-α: (144.807 ± 1.348) pg/mL] and the infection and high-fat diet group [IL-1β: (110.131 ± 12.946) pg/mL; TNF-α: (131.764 ± 27.831) pg/mL] (all P values < 0.05). In addition, lower CYP 4A1 mRNA (3.00 ± 0.81) and protein expression (0.40 ± 0.02) was quantified in lung specimens in the infection and normal diet group than in the normal control group [(5.03 ± 2.05) and (0.84 ± 0.14)] and the infection and high-fat diet group [(11.19 ± 3.51) and (0.68 ± 0.18)] (all P values < 0.05). High-fat intake may alleviate lung injuries caused by P. proliferus infection in rats through up-regulating CYP 4A1 expression in lung tissues at both translational and transcriptional levels.

A Longitudinal Study of the Relationship of Adiponectin with Reproduction in Infertile Women Undergoing IVF/ICSI Treatment, and an Experimental Study in Human Granulosa Cells.

This study investigated the roles of adiponectin in IVF treatment during Phase I (the basal stage before gonadotropin administration), Phase II (approximately 8 days after gonadotropin administration), and Phase III (on the ovum pick-up day), as well as the effects of adiponectin on CYP19A1 and the FSH receptor (FSHR) mRNA expression in a human granulosa-like tumor cell line (KGN). In human subjects (a longitudinal study, n = 30), blood samples were collected in all phases, while follicular fluid (FF) was only collected in Phase III. The participants were classified into successful and unsuccessful groups based on the determination of fetal heartbeats. KGN cells were treated with adiponectin/FSH/IGF-1 (an experimental study, n = 3). There was no difference in the adiponectin levels between successful and unsuccessful pregnancies in the FF (Phase III) and in serum (all phases), as well as among the three phases in both groups. Serum FSH (Phase I) was positively associated with serum adiponectin in the unsuccessful group, but it had a negative association in the successful group (all phases). Serum adiponectin and serum FSH (Phase I) were positively correlated in the unsuccessful group, whereas they were negatively correlated (all phases) in the successful group. The serum adiponectin levels (Phase III) were significantly higher than in the FF in unsuccessful pregnancies, but there was no difference in successful pregnancies. FF adiponectin concentrations were negatively correlated with serum LH in successful subjects. In KGN cells, adiponectin had no influence on CYP19A1 and FSHR mRNA expression. High adiponectin levels in serum compared to FF (Phase III) in unsuccessful subjects might negatively impact IVF treatment.

Attenuation of phenobarbital-induced cytochrome P450 expression in carbon tetrachloride-induced hepatitis in mice models.

Certain pathological conditions, such as inflammation, are known to affect basal cytochrome P450 (CYP) expression by modulating transcriptional regulation, and the pharmacokinetics of drugs can vary among patients. However, changes in drug-induced CYP expression under pathological conditions have not been elucidated in detail. Here, we investigated the effects of hepatic inflammation and injury on phenobarbital-induced expression of CYP isoforms in mice. Phenobarbital was administered once as a CYP inducer in the carbon tetrachloride-induced hepatitis model mice. The mRNA expression levels of Cyp3a11 and Cyp2b10 in the liver and small intestine were measured using reverse transcription polymerase chain reaction. The enzymatic activity of CYP3A in liver S9 was evaluated using midazolam as the substrate. Phenobarbital increased the mRNA expression of Cyp3a11 and Cyp2b10 in the liver of healthy mice, but not in the small intestine. Increased mRNA expression of hepatic Cyp3a11 and Cyp2b10 by phenobarbital was significantly suppressed in the hepatitis model mice. Hepatitis also suppressed the increased CYP3A enzymatic activity induced by phenobarbital in liver S9, consistent with the results of Cyp3a11 mRNA expression. These results suggest that the inducibility of CYP by phenobarbital may vary in patients with hepatitis, indicating that pharmacokinetic drug-drug interactions can be altered under certain pathological conditions.

Cyp26a1 supports postnatal retinoic acid homeostasis and glucoregulatory control

Considerable evidence confirms the importance of Cyp26a1 to all-trans-retinoic acid (RA) homeostasis during embryogenesis. In contrast, despite its presence in postnatal liver as a potential major RA catabolizing enzyme and its acute sensitivity to induction by RA, some data suggested that Cyp26a1 contributes only marginally to endogenous RA homeostasis postnatally. We report reevaluation of a conditional Cyp26a1 knockdown in the postnatal mouse. The current results show that Cyp26a1 mRNA in WT mouse liver increases 16-fold upon refeeding after a fast, accompanied by an increased rate of RA elimination and a 41% decrease in the RA concentration. In contrast, Cyp26a1 mRNA in the refed homozygotic knockdown reached only 2% of its extent in WT during refeeding, accompanied by a slower rate of RA catabolism and no decrease in liver RA, relative to fasting. Refed homozygous knockdown mice also had decreased Akt1 and 2 phosphorylation and pyruvate dehydrogenase kinase 4 (Pdk4) mRNA and increased glucokinase (Gck) mRNA, glycogen phosphorylase (Pygl) phosphorylation, and serum glucose, relative to WT. Fasted homozygous knockdown mice had increased glucagon/insulin relative to WT. These data indicate that Cyp26a1 participates prominently in moderating the postnatal liver concentration of endogenous RA and contributes essentially to glucoregulatory control.

Modulation of TCDD induced Cyp1a1 mRNA by the Co-repressor SMRT

Modulation of TCDD induced Cyp1a1 mRNA by the Co-repressor SMRT

3D Spheroid Primary Human Hepatocytes for Prediction of Cytochrome P450 and Drug Transporter Induction.

Primary human hepatocytes (PHHs) have been the gold standard in vitro model for the human liver and are crucial to predict hepatic drug-drug interactions. The aim of this work was to assess the utility of 3D spheroid PHHs to study induction of important cytochrome P450 (CYP) enzymes and drug transporters. 3D spheroid PHHs from three different donors were treated for four days with rifampicin, dicloxacillin, flucloxacillin, phenobarbital, carbamazepine, efavirenz, omeprazole or β-naphthoflavone. Induction of CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and transporters P-glycoprotein (P-gp)/ABCB1, multidrug resistance-associated protein 2 (MRP2)/ABCC2, ABCG2, organic cation transporter 1 (OCT1)/SLC22A1, SLC22A7, SLCO1B1 and SLCO1B3 were evaluated at mRNA and protein levels. Enzyme activity of CYP3A4, CYP2B6, CYP2C19 and CYP2D6 were also assessed. Induction of CYP3A4 protein and mRNA correlated well for all donors and compounds and had a maximal induction of 5- to 6-fold for rifampicin, which closely correlates to induction observed in clinical studies. Rifampicin induced the mRNA of CYP2B6 and CYP2C8 by 9- and 12-fold, while the protein levels of these CYPs reached 2- and 3-fold induction, respectively. Rifampicin induced CYP2C9 protein by 1.4-fold, while the induction of CYP2C9 mRNA was over 2-fold in all donors. Rifampicin induced ABCB1, ABCC2 and ABCG2 by 2-fold. In conclusion, 3D spheroid PHHs is a valid model to investigate mRNA and protein induction of hepatic drug-metabolizing enzymes and transporters, and this model provides a solid basis to study induction of CYPs and transporters, which translates to clinical relevance.

Characterization of ovarian follicles, serum steroid hormone concentration, and steroidogenic gene expression profiles in the developing ovarian follicles in White King pigeons

Paired pigeons only lay 2 eggs in a laying period, which is closely related to ovarian follicle development, but this process is not well understood. In this study, 60 pairs of 12-mo-old White King pigeons were selected and serum and follicles were collected at 4 stages of laying interval (LI), including the first (LI1), the third (LI3), the fifth (LI5), and the seventh day (LI7). Morphological results showed that paired pigeons normally had 2 preovulatory follicles and the second-largest follicle (F2) developed from LI3 and had been selected in LI5. Prehierarchical follicles were coupled and hierarchical, which was in accordance with its clutch size. The P4 concentration increased gradually from LI1 to LI5, reaching a maximum of 30.67 ng/mL in LI5 and decreasing to 27.83 ng/mL in LI7 (P < 0.05). The levels of T in LI1 and LI5 were higher than LI3 and LI7 (P < 0.05), although there was no significant difference in E2 in LI (P > 0.05), but it stayed at high levels. In the TCs of the largest follicle (F1), HSD3B1 mRNA and HSD17B1 mRNA levels peaked in LI7. The expression pattern of CYP17A1 and CYP19A1 was similar, increasing from LI3 to LI5 and then decreasing. In the TCs of F2, the expressions of HSD3B1 and CYP17A1 had no significant difference between LI5 and LI7 (P > 0.05), while the expression pattern of HSD17B1 and CYP19A1 was the opposite. In TCs of SF1, HSD3B1 mRNA level peaked in LI3 while CYP19A1 mRNA levels peaked in LI7. The expression of CYP17A1 had a minor change (P > 0.05) and the expression pattern of HSD17B1 was similar to F1. It was concluded that the morphological characteristics of follicles during the LI for the first time, including the number and diameter of small follicles (SFs) and hierarchical follicles in pigeon and the concentrations of steroid hormones and expressions of steroidogenic genes in TCs of different follicles could explain the growth and selection of 2 preovulatory follicles. This study facilitates further research into the regulation of ovulation and egg production in pigeons.

Nrf2-/- regulated lung DNA demethylation and CYP2E1 DNA methylation under PM2.5 exposure.

Cytochrome P450 (CYP450) can mediate fine particulate matter (PM2.5) exposure leading to lung injury. Nuclear factor E2-related factor 2 (Nrf2) can regulate CYP450 expression; however, the mechanism by which Nrf2-/- (KO) regulates CYP450 expression via methylation of its promoter after PM2.5 exposure remains unclear. Here, Nrf2-/- (KO) mice and wild-type (WT) were placed in a PM2.5 exposure chamber (PM) or a filtered air chamber (FA) for 12weeks using the real-ambient exposure system. The CYP2E1 expression trends were opposite between the WT and KO mice following PM2.5 exposure. After exposure to PM2.5, CYP2E1 mRNA and protein levels were increased in WT mice but decreased in KO mice, and CYP1A1 expression was increased after exposure to PM2.5 in both WT and KO mice. CYP2S1 expression decreased after exposure to PM2.5 in both the WT and KO groups. We studied the effect of PM2.5 exposure on CYP450 promoter methylation and global methylation levels in WT and KO mice. In WT and KO mice in the PM2.5 exposure chamber, among the methylation sites examined in the CYP2E1 promoter, the CpG2 methylation level showed an opposite trend with CYP2E1 mRNA expression. The same relationship was evident between CpG3 unit methylation in the CYP1A1 promoter and CYP1A1 mRNA expression, and between CpG1 unit methylation in the CYP2S1 promoter and CYP2S1 mRNA expression. This data suggests that methylation of these CpG units regulates the expression of the corresponding gene. After exposure to PM2.5, the expression of the DNA methylation markers ten-eleven translocation 3 (TET3) and 5-hydroxymethylcytosine (5hmC) was decreased in the WT group but significantly increased in the KO group. In summary, the changes in CYP2E1, CYP1A1, and CYP2S1 expression in the PM2.5 exposure chamber of WT and Nrf2-/- mice might be related to the specific methylation patterns in their promoter CpG units. After exposure to PM2.5, Nrf2 might regulate CYP2E1 expression by affecting CpG2 unit methylation and induce DNA demethylation via TET3 expression. Our study revealed the underlying mechanism for Nrf2 to regulate epigenetics after lung exposure to PM2.5.

Ginsenoside Rb1 for overcoming cisplatin-insensitivity of A549/DDP cells in vitro and vivo through the dual-inhibition on two efflux pumps of ABCB1 and PTCH1

BackgroundThe multi-drug resistance is an inherent weakness in the chemotherapeutics of non-small cell lung cancer occurring frequently all over the world. Clinically, ginseng and Chinese medicinal prescriptions including ginseng usually used as anti-tumor adjuncts due to its characteristic of qi-invigorating, which could improve the curative effect of chemotherapy drugs and reduce their toxic side effects. Triterpenoid saponins are the crucial active ingredients in Panax ginseng, and Ginsenoside Rb1 is of the highest quantities. However, the research on the tumor drug-resistance reversal effect and mechanism of ginsenoside Rb1 is still not clear. PurposeThis study aimed to systematically estimate the reversal activity of Ginsenoside Rb1 on cisplatin-insensitivity of A549/DDP cells and to reveal its prospective molecular mechanism. MethodsMTT assay were conducted to evaluate the reversal activity on cisplatin-insensitivity of A549/DDP cells of Ginsenoside Rb1in vitro, and the behavior was also studied by establishing a subcutaneous transplanted tumor model of A549/DDP in BALB/c-nu mice. In addition, P-gp ATPase activity assay, cisplatin accumulation assay, Annexin V-FITC apoptosis assay, real-time qPCR analysis and western blotting analysis were used to clarify the potential mechanism. ResultsGinsenoside Rb1 could effectively reverse the cisplatin-resistance of A549/DDP in vitro and vivo. And after the co-treatment of Ginsenoside Rb1 plus cisplatin, the accumulation of cisplatin increased in A549/DDP cells, which was accompanied with the down-regulation of the mRNA and protein expression levels of ABCB1, SHH, PTCH1 and GLI2. Besides, the apoptosis-inducing ability of cisplatin improved by the relative regulation on the protein expression level of Bax and Bcl-2. Far more importantly, the changes of CYP3A4 mRNA and protein levels were not significant. ConclusionGinsenoside Rb1 could increase the concentration of intracellular cisplatin and improve the insensitivity for cisplatin on A549/DDP cells. Even better, there was perhaps no unpredictable CYP3A4-mediated pharmacokinetic interactions after the combination of Ginsenoside Rb1 plus cisplatin. Ginsenoside Rb1 was a probable reversal agent for the cisplatin-insensitivity of A549/DDP cells, with a bifunction of inhibiting the efflux of two drug pumps (P-gp and PTCH1) by targeting ABCB1 and Hedgehog (Hh) pathway. In general, this research laid the groundwork for the development of a new reversal agent for the cisplatin-insensitivity of NSCLC.

Modulation of cytochrome P450 1A (CYP1A) enzymes by monomethylmonothioarsonic acid (MMMTAV) in vivo and in vitro

Inorganic arsenic (iAs) is a natural toxicant which, upon entering the biosphere, undergoes extensive biotransformation and becomes a portal for generating various organic intermediates/products. The chemical diversity of iAs-derived organoarsenicals (oAs) is accompanied by varying degree of toxicity that can be held responsible, at least partly, for the overall health outcome of the originally encountered parent inorganic molecule. Such toxicity may originate from arsenicals ability to modulate cytochrome P450 1A (CYP1A) enzymes, whose activity is critical in activating/detoxifying procarcinogens. In this study, we evaluated the effect of monomethylmonothioarsonic acid (MMMTAV) on CYP1A1 and CYP1A2 in absence and presence of their inducer; 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Therefore, C57BL/6 mice were intraperitoneally injected with 12.5 mg/kg MMMTAV, with or without 15 μg/kg TCDD for 6 and 24 h. Moreover, murine Hepa-1c1c7 and human HepG2 cells were treated with MMMTAV (1, 5, and 10 μM), with or without 1 nM TCDD for 6 and 24 h. MMMTAV significantly inhibited TCDD-mediated induction of CYP1A1 mRNA, both in vivo and in vitro. This effect was attributed to decreased transcriptional activation of CYP1A regulatory element. Interestingly, MMMTAV significantly increased TCDD-induced CYP1A1 protein and activity in C57BL/6 mice and Hepa-1c1c7 cells, while both were significantly inhibited by MMMTAV treatment in HepG2 cells. CYP1A2 mRNA, protein and activity induced by TCDD were significantly increased by MMMTAV co-exposure. MMMTAV had no effect on CYP1A1 mRNA stability or protein stability and did not alter their half-lives. At basal level, only CYP1A1 mRNA was significantly decreased in MMMTAV-treated Hepa-1c1c7 cells. Our findings show that MMMTAV exposure potentiates procarcinogen-induced catalytic activity of both CYP1A1 and CYP1A2 in vivo. This effect entails excessive activation of such procarcinogens upon co-exposure, with potentially negative health-related outcomes.

Liver microsomal cytochrome P450 3A-dependent drug oxidation activities in individual dogs

Drug oxidations are mediated mainly by cytochromes P450 (P450s or CYPs). CYP3As are an important P450 subfamily and include liver-specific CYP3A12 and intestine-specific CYP3A98 in dogs. Individual differences in drug oxidation activities were investigated, including correlations with immunoreactive CYP3A protein intensities and CYP3A mRNA expression levels in livers. Pooled and individual dog liver microsomes showed activities towards nifedipine, midazolam, alprazolam, and estradiol, but the levels of catalytic activities varied approximately twofold among the individual dogs. One dog harboured a CYP1A2 variant causing protein deletion but showed higher activities than the other dogs towards nifedipine oxidation, midazolam 1′-hydroxylation, alprazolam 4-hydroxylation, estradiol 16α-hydroxylation activities, and caffeine C 8-hydroxylation; the latter is used as a reference reaction for CYP1A. In individual dog liver microsomes, the intensities of the immunochemical bands with anti-human CYP3A4 and anti-rat CYP3A2 antibodies along with CYP3A12 and CYP3A26 mRNA expression levels showed good correlations (p < 0.05) with nifedipine oxidation, midazolam 1′- and 4-hydroxylation, alprazolam 1′- and 4-hydroxylation, and estradiol 16α-hydroxylation activities. These results suggest that the oxidation activities of dog liver microsomes towards nifedipine and other typical CYP3A-catalyzed drugs exhibit approximately twofold individual differences and were predominantly mediated by liver-specific CYP3A12 in the dogs.

Mitoguardin2 Is Associated With Hyperandrogenism and Regulates Steroidogenesis in Human Ovarian Granulosa Cells.

Polycystic ovary syndrome (PCOS) is an endocrinopathy characterized by hyperandrogenism, anovulation, and polycystic ovaries, in which hyperandrogenism manifests by excess androgen and other steroid hormone abnormalities. Mitochondrial fusion is essential in steroidogenesis, while the role of mitochondrial fusion in granulosa cells of hyperandrogenic PCOS patients remains unclear. In this study, mRNA expression of mitochondrial fusion genes mitoguardin1, -2 (MIGA 1, -2) was significantly increased in granulosa cells of hyperandrogenic PCOS but not PCOS with normal androgen levels, their mRNA expression positively correlated with testosterone levels. Dihydrotestosterone (DHT) treatment in mice led to high expression of MIGA2 in granulosa cells of ovulating follicles. Testosterone or forskolin/ phorbol 12-myristate 13-acetate treatments increased expression of MIGA2 and the steroidogenic acute regulatory protein (StAR) in KGN cells. MIGA2 interacted with StAR and induced StAR localization on mitochondria. Furthermore, MIGA2 overexpression significantly increased cAMP-activated protein kinase A (PKA) and phosphorylation of AMP-activated protein kinase (pAMPK) at T172 but inhibited StAR protein expression. However, MIGA2 overexpression increased CYP11A1, HSD3B2, and CYP19A1 mRNA expression. As a result, MIGA2 overexpression decreased progesterone but increased estradiol synthesis. Besides the androgen receptor, testosterone or DHT might also regulate MIGA2 and pAMPK (T172) through LH/choriogonadotropin receptor-mediated PKA signaling. Taken together, these findings indicate that testosterone regulates MIGA2 via PKA/AMP-activated protein kinase signaling in ovarian granulosa cells. It is suggested mitochondrial fusion in ovarian granulosa cells is associated with hyperandrogenism and potentially leads to abnormal steroidogenesis in PCOS.

Boscalid shows increased thyroxin-glucuronidation in rat but not in human hepatocytes in vitro.

The fungicide boscalid induces thyroid histopathological and hormone effects in the rat, secondary to liver enzyme induction. To assess the human relevance of liver enzyme induction presumably leading to thyroid hormone disruption, a species comparative in vitro study on T4-glucuronidation was conducted. Currently, no guidelines how to evaluate Phase II induction are in place. Therefore, we investigated the optimal conditions to evaluate Phase I and Phase II induction potential of boscalid in primary rat (PRH) and human (PHH) hepatocytes. Endpoints included mRNA gene expression and enzyme activities (cytochrome P450 isozymes [CYPs] and uridine diphosphate-glucuronosyltransferases [UGTs]), measured after 3 (D3) and 7 (D7) days of exposure to reference compounds and to 5, 10, and 20 μM boscalid, focusing on T4-glucuronidation. Basal CYP activities and T4 glucuronidation were similar or higher on D7 than D3. The highest induction responses of CYPs were on D3, whereas UGT induction and T4-glucuronidation increases were highest on D7. Boscalid induced CYP1A, CYP2B, and CYP3A mRNA and/or increased related activities in PRH and PHH. Species differences in the induction pattern of UGT genes by reference inducers (ß-naphthoflavone [BNF], 5-pregnen-3ß-ol-20-one-16α-carbonitirile [PCN], rifampicin [RIF], and phenobarbital [PB]) and boscalid were seen: UGT1A1, UGT1A3, and UGT1A9 were predominantly induced in PHH, while UGT2B1 was predominantly induced in PRH. Basal activity levels for T4-glucuronidation were very low in humans and an order of magnitude higher in rat, for this reason increases in activities were assessed as delta activity to the control. Significant increases in T4-glucuronidation occurred with boscalid in rat but not in human hepatocytes.