All-trans retinoic acid regulates hepatic bile acid homeostasis

Retinoic acid (RA) and bile acids share common roles in regulating lipid homeostasis and insulin sensitivity. In addition, the receptor for RA (retinoid x receptor) is a permissive partner of the receptor for bile acids, farnesoid x receptor (FXR/NR1H4). Thus, RA can activate the FXR-mediated pathway as well. The current study was designed to understand the effect of all-trans RA on bile acid homeostasis. Mice were fed an all-trans RA-supplemented diet and the expression of 46 genes that participate in regulating bile acid homeostasis was studied. The data showed that all-trans RA has a profound effect in regulating genes involved in synthesis and transport of bile acids. All-trans RA treatment reduced the gene expression levels of Cyp7a1, Cyp8b1, and Akr1d1, which are involved in bile acid synthesis. All-trans RA also decreased the hepatic mRNA levels of Lrh-1 (Nr5a2) and Hnf4α (Nr2a1), which positively regulate the gene expression of Cyp7a1 and Cyp8b1. Moreover, all-trans RA induced the gene expression levels of negative regulators of bile acid synthesis including hepatic Fgfr4, Fxr, and Shp (Nr0b2) as well as ileal Fgf15. All-trans RA also decreased the expression of Abcb11 and Slc51b, which have a role in bile acid transport. Consistently, all-trans RA reduced hepatic bile acid levels and the ratio of CA/CDCA, as demonstrated by liquid chromatography-mass spectrometry. The data suggest that all-trans RA-induced SHP may contribute to the inhibition of CYP7A1 and CYP8B1, which in turn reduces bile acid synthesis and affects lipid absorption in the gastrointestinal tract.     

1,10-Phenanthroline stabilizes mRNA of the carcinogen-metabolizing enzyme,cytochrome P450 1a1

1,10-phenanthroline (phen), flufenamic acid, and indomethacin are inhibitors of aldo-keto reductases 1C1 (AKR1C1), but only phen decreased the benzo[a]pyrene (BaP)-induced cytochrome P450 1a1 (Cyp1a1) protein level. Therefore the decrease in the BaP-induced Cyp1a1 protein level was not due to inhibition of Akr1c1, but to phen itself. Phen decreased the BaP-induced Cyp1a1 promoter activity and protein expression, and in contrast, it increased Cyp1a1 mRNA, resulting from an increase in mRNA stability. Phen is also known as a transition metal ion-chelator. Along with the phen study, we also found that Zn²⁺, Fe²⁺ and Cu²⁺ increased Cyp1a1 mRNA and protein stability. Our results show that phen stabilized the mRNA of Cyp1a1, although it decreased cell viability. In addition, Zn²⁺ and Fe²⁺ highly neutralized phen's suppression of Cyp1a1 protein expression, but they only slightly neutralized phen's promotion of mRNA stability and suppression of cell viability, and had no effect on phen's suppression of promoter activity. Phen's effect on Cyp1a1 expression was reversible, which indicates that phen is non-covalently linked to its target. This report elucidates a new role for phen of stabilizing Cyp1a1 mRNA, and provides information for further studies on mRNA stabilization.     

Evidence of the Involvement of a Cyclase Gene in the Biosynthesis of Ochratoxin A in Aspergillus carbonarius

Ochratoxin A (OTA) is a well-known mycotoxin with wide distribution in food and feed. Fungal genome sequencing has great utility for identifying secondary metabolites gene clusters for known and novel compounds. A comparative analysis of the OTA-biosynthetic cluster in A. steynii, A. westerdijkiae, A. niger, A. carbonarius, and P. nordicum has revealed a high synteny in OTA cluster organization in five structural genes (otaA, otaB, ota, otaR1, and otaD). Moreover, a recent detailed comparative genome analysis of Aspergilli OTA producers led to the identification of a cyclase gene, otaY, located in the OTA cluster between the otaA and otaB genes, encoding for a predicted protein with high similarity to SnoaLs domain. These proteins have been shown to catalyze ring closure steps in the biosynthesis of polyketide antibiotics produced in Streptomyces. In the present study, we demonstrated an upregulation of the cyclase gene in A. carbonarius under OTA permissive conditions, consistent with the expression trends of the other OTA cluster genes and their role in OTA biosynthesis by complete gene deletion. Our results pointed out the involvement of a cyclase gene in OTA biosynthetic pathway for the first time. They represent a step forward in the understanding of the molecular basis of OTA biosynthesis in A. carbonarius.     

Effects of valproate,an HDAC inhibitor,on the expression of folate carriers and folate metabolism-related genes in the placenta of rats

Valproate (VPA), an antiepileptic drug, is known to inhibit histone deacetylases (HDACs). Exposure to VPA during pregnancy increases several fetal risks. The maintenance of folate level during pregnancy is essential for adequate fetal development, and the placenta plays a critical role in supplying nutrients to the fetus. The aim of this study was to elucidate the effects of VPA on the gene expression of folate carriers and metabolizing enzymes in the rat placenta at both mid and late gestation periods. Pregnant rats were orally administered VPA on a single day or 4 days (repeated administration). Gene expression of folate carriers (Folr1, Slc19a1, Slc46a1) and metabolizing enzymes (Cth, Mtr, Mtrr, Mthfr, Dhfr) was assessed in the placenta on gestational day (GD) 13 or GD20. In the control rats, the expression of Folr1, Slc46a1, Cth, and Mthfr tended to be upregulated, whereas that of Mtrr and Dhfr was downregulated during gestation; the expression of Slc19a1 and Mtr did not change. Repeated VPA administration reduced the placental expression of Folr1and Mtr on GD20 and increased the expression of Dhfr on GD13 compared with the control. These findings indicate that administration of VPA alters the placental gene expression of folate carriers and metabolism-related enzymes.     

Epigenetic regulation of developmental expression of Cyp2d genes in mouse liver

CYP2D6 expression in liver is age-dependent. Because epigenetic mechanisms, such as DNA methylation and histone modifications, modulate age-related gene expression during development, and are highly conserved among species, the current study examined the epigenetic regulation of age-related expression of the Cyp2d genes in mouse liver. DNA methylation (DNAme), histone 3 lysine 4 dimethylation (H3K4me2), and histone 3 lysine 27 trimethylation (H3K27me3) was established by ChIP-on-chip tiling microarrays from mouse livers at prenatal, neonatal, and adult stages. Levels of DNAme, H3K4me2, and H3K27me3 were analyzed in a genomic region containing the Cyp2d clustering genes and their surrounding genes. Gradually increased expression levels of the Cyp2d9, Cyp2d10, Cyp2d22, and Cyp2d26 genes from prenatal, through neonatal, to adult are associated with gradually increased levels of H3K4me2 in the nucleosomes associated with these genes. Gene expression patterns during liver development in several Cyp2d surrounding genes, such as Srebf2, Sept3, Ndufa6, Tcf2, Nfam1, and Cyb5r3, could be also explained by changes of DNA methylation, H3K4me2, or H3K27me3 in those genes. In conclusion, the current study demonstrates that the changes of DNA methylation and histone modifications are associated with age-related expression patterns of the Cyp2d genes and their surrounding genes in liver cells during development.     

Effects of aqueous extract of Ruta graveolens and its ingredients on cytochrome P450, uridine diphosphate (UDP)-glucuronosyltransferase,and reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H)-quinone oxidoreductase in mice

Ruta graveolens (the common rue) has been used for various therapeutic purposes, including relief of rheumatism and treatment of circulatory disorder. To elucidate the effects of rue on main drug-metabolizing enzymes, effects of an aqueous extract of the aerial part of rue and its ingredients on cytochrome P450 (P450/CYP), uridine diphosphate (UDP)-glucuronosyltransferase, and reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H):quinone oxidoreductase were studied in C57BL/6JNarl mice. Oral administration of rue extract to males increased hepatic Cyp1a and Cyp2b activities in a dose-dependent manner. Under a 7-day treatment regimen, rue extract (0.5 g/kg) induced hepatic Cyp1a and Cyp2b activities and protein levels in males and females. This treatment increased hepatic UDP-glucuronosyltransferase activity only in males. However, NAD(P)H:quinone oxidoreductase activity remained unchanged. Based on the contents of rutin and furanocoumarins of mouse dose of rue extract, rutin increased hepatic Cyp1a activity and the mixture of furanocoumarins (F_mix) increased Cyp2b activities in males. The mixture of rutin and F_mix increased Cyp1a and Cyp2b activities. These results revealed that rutin and F_mix contributed at least in part to the P450 induction by rue.     

Methoxychlor reduces estradiol levels by altering steroidogenesis and metabolism in mouse antral follicles in vitro

The organochlorine pesticide methoxychlor (MXC) is a known endocrine disruptor that affects adult rodent females by causing reduced fertility, persistent estrus, and ovarian atrophy. Since MXC is also known to target antral follicles, the major producer of sex steroids in the ovary, the present study was designed to test the hypothesis that MXC decreases estradiol (E₂) levels by altering steroidogenic and metabolic enzymes in the antral follicles. To test this hypothesis, antral follicles were isolated from CD-1 mouse ovaries and cultured with either dimethylsulfoxide (DMSO) or MXC. Follicle growth was measured every 24 h for 96 h. In addition, sex steroid hormone levels were measured using enzyme-linked immunosorbent assays (ELISA) and mRNA expression levels of steroidogenic enzymes as well as the E₂ metabolic enzyme Cyp1b1 were measured using qPCR. The results indicate that MXC decreased E₂, testosterone, androstenedione, and progesterone (P₄) levels compared to DMSO. In addition, MXC decreased expression of aromatase (Cyp19a1), 17β-hydroxysteroid dehydrogenase 1 (Hsd17b1), 17α-hydroxylase/17,20-lyase (Cyp17a1), 3β hydroxysteroid dehydrogenase 1 (Hsd3b1), cholesterol side-chain cleavage (Cyp11a1), steroid acute regulatory protein (Star), and increased expression of Cyp1b1 enzyme levels. Thus, these data suggest that MXC decreases steroidogenic enzyme levels, increases metabolic enzyme expression and this in turn leads to decreased sex steroid hormone levels.     

Evaluation of a bioluminescent mouse model expressing aromatase PII-promoter-controlled luciferase as a tool for the study of endocrine disrupting chemicals

Dysfunction of the enzyme aromatase (CYP19) is associated with endocrine pathologies such as osteoporosis, impaired fertility and development of hormone-dependent cancers. Certain endocrine disrupting chemicals affect aromatase expression and activity in vitro, but little is known about their ability to do so in vivo. We evaluated a bioluminescent mouse model (LPTA®)CD-1-Tg(Cyp19-luc)-Xen) expressing luciferase under control of the gonadal aromatase pII promoter as an in vivo screening tool for chemicals that may affect aromatase expression. We studied the effects of forskolin, pregnant mare serum gonadotropin and atrazine in this model (atrazine was previously shown to induced pII-promoter-driven aromatase expression in H295R human adrenocortical carcinoma cells). About 2-4 out of every group of 10 male or female Cyp19-luc mice injected i.p. with 10 mg/kg forskolin had increased gonadal bioluminescence after 3-5 days compared to controls; the others appeared non-responsive. Similarly, about 4 per group of 9 individual females injected with pregnant mare serum gonadotropin had increased ovarian bioluminescence after 24 h. There was a statistically significant correlation between ovarian bioluminescence and plasma estradiol concentrations (n = 14; p = 0.022). Males exposed to a single dose of 100 mg/kg or males and females exposed to 5 daily injections of 30 mg/kg atrazine showed no change in gonadal bioluminescence over a 7 day period, but a significant interaction was found between atrazine (100 mg/kg) and time in female mice (p < 0.05; two-way ANOVA). Ex vivo luciferase activity in dissected organs was increased by forskolin in testis, epididymis and ovaries. Atrazine (30 mg/kg/day) increased (30%) luciferase activity significantly in epididymis only. In conclusion, certain individual Cyp19-luc mice are highly responsive to aromatase inducers, suggesting this model, with further optimization, may have potential as an in vivo screening tool for environmental contaminants.     

Involvement of oxidative stress in hepatocellular tumor-promoting activity of oxfendazole in rats

The tumor-promoting effects of oxfendazole (OX), a benzimidazole anthelmintic, were investigated using a medium-term rat hepatocarcinogenesis model. Six-week-old male F344 rats received an intraperitoneal injection of N-diethylnitrosamine (DEN) and were given a powdered diet containing 0 or 500 ppm OX for 6 weeks from 2 weeks after DEN treatment. All animals were subjected to two-thirds partial hepatectomy 1 week after OX treatment. The numbers and areas of glutathione S-transferase placental form (GST-P)-positive foci were significantly increased in the livers of rats treated with OX, with concomitantly increased cell proliferation, compared with those in the livers of the DEN alone group. Quantitative real-time RT-PCR analysis revealed that OX induced not only mRNA expression of phase I enzymes Cyp1a1, Cyp1a2, but also Nrf2-regulated phase II enzymes such as Gpx2, Nqo1, Yc2, Akr7a3 and Gstm1, presumably due to an adaptive response against OX-induced oxidative stress. Reactive oxygen species production increased in microsomes isolated from the livers of OX-treated rats. Furthermore, OX enhanced oxidative DNA damage (as assessed by 8-hydroxydeoxyguanosine; 8-OHdG) and lipid peroxidation (as assessed by thiobarbituric acid-reactive substances; TBARS). These results suggest that administration of OX at a high dose and for a long term enhances oxidative stress responses, which may contribute to its tumor-promoting potential in rats.     

Diethylstilbestrol at environmental levels affects the development of early life stage and target gene expression in Japanese Medaka (<Emphasis Type="Italic">Oryzias latipes</Emphasis>)

In this study, the biologic effects of DES on the early life and adult life stages of Japanese medaka (Oryzias latipes) were evaluated. At the early life stage, the fertilized eggs were exposed to 1–1000 ng/L diethylstilbestrol (DES) for 15 days and the hatched larvae were continually exposed to the same concentrations for an additional 25 days. Significant adverse effects on hatchability, time to hatching and mortality rate occurred at DES concentrations of 100 and 1000 ng/L, while the abnormality (scoliosis and abdominal swelling) rate was significantly increased at 10 ng/L and above. After exposure, the fish were maintained in charcoal-dechlorinated tap water for a further 30 days. Only the male gonadosomatic index (GSI) at 1000 ng/L was significantly increased. At concentrations greater than 1 ng/L, estrogen receptor α (ERα) mRNA in both sexes and vitellogenin-I (Vtg-I) mRNA in males were significantly down-regulated; while Vtg-I mRNA in females was significantly up-regulated. When sexually mature medaka were exposed to 10 and 1000 ng/L DES for 21 days, only the GSI in females was significantly decreased at 1000 ng/L. At 10 and 1000 ng/L, ERα mRNA in both sexes was significantly down-regulated, while Vtg-I mRNA in males was significantly up-regulated. These findings showed that DES at the environmental concentration of 10 ng/L can affect the early life stage development of medaka and alter liver ERα and Vtg-I gene expression. Therefore, if we only focused on these sensitive toxicity endpoints such as ERα and Vtg-I mRNA expression, DES has a strong estrogenic effect on Japanese medaka.     

Sex- and age-dependent effects of maternal organophosphate flame-retardant exposure on neonatal hypothalamic and hepatic gene expression

After the phase-out of polybrominated diphenyl ethers, their replacement compounds, organophosphate flame retardants (OPFRs) became ubiquitous in home and work environments. OPFRs, which may act as endocrine disruptors, are detectable in human urine, breast milk, and blood samples collected from pregnant women. However, the effects of perinatal OPFR exposure on offspring homeostasis and gene expression remain largely underexplored. To address this knowledge gap, virgin female mice were mated and dosed with either a sesame oil vehicle or an OPFR mixture (tris(1,3-dichloro-2-propyl)phosphate, tricresyl phosphate, and triphenyl phosphate, 1 mg/kg each) from gestational day (GD) 7 to postnatal day (PND) 14. Hypothalamic and hepatic tissues were collected from one female and one male pup per litter on PND 0 and PND 14. Expression of genes involved in energy homeostasis, reproduction, glucose metabolism, and xenobiotic metabolism were analyzed using quantitative real-time PCR. In the mediobasal hypothalamus, OPFR increased Pdyn, Tac2, Esr1, and Pparg in PND 14 females. In the liver, OPFR increased Pparg and suppressed Insr, G6pc, and Fasn in PND 14 males and increased Esr1, Foxo1, Dgat2, Fasn, and Cyb2b10 in PND 14 females. We also observed striking sex differences in gene expression that were dependent on the age of the pup. Collectively, these data suggest that maternal OPFR exposure alters hypothalamic and hepatic development by influencing neonatal gene expression in a sex-dependent manner. The long-lasting consequences of these changes in expression may disrupt puberty, hormone sensitivity, and metabolism of glucose, fatty acids, and triglycerides in the maturing juvenile.     

Urinary bisphenol A concentrations and cytochrome P450 19 A1 (Cyp19) gene expression in ovarian granulosa cells: An in vivo human study

BackgroundExposure to bisphenol A (BPA), a chemical widely used in consumer products, has been associated with in vitro Cyp19 gene expression.ObjectiveTo evaluate an in vivo human model of Cyp19 gene expression in granulosa cells.Study DesignA subset of an ongoing prospective cohort study of women undergoing in vitro fertilization (IVF) at Massachusetts General Hospital.MethodsMixed effect models were used to evaluate the association of urinary BPA concentrations with granulosa cell Cyp19 mRNA expression.ResultsIn 61 women undergoing 76 IVF cycles, adjusted changes in mean Cyp19 expression (β estimate (95% CI)) for quartiles 2, 3 and 4 as compared to the lowest quartile were: −0.97 (−2.22, 0.28); −0.97 (−2.18, 0.24) and −0.38 (−1.58, 0.82).ConclusionsAn in vivo model for evaluation of Cyp19 gene expression was developed for use in epidemiologic studies. In this pilot study, we found no statistically significant linear association between urinary BPA concentrations and Cyp19 expression.