Di (2-ethyl-hexyl) phthalate disrupts placental growth in a dual blocking mode.

Di (2-ethyl-hexyl) phthalate (DEHP) is a widely used plasticizer. Maternal DEHP exposure inhibits cell proliferation and reduces placentas size, which associates with fetal growth restriction and adulthood diseases. However, the mechanism of placental cell proliferation inhibition by DEHP remains elusive. This study investigated the effect of DEHP on placental cell proliferation from cell cycle arrest. Utilizing in vitro and in vivo experiments, we investigated cell cycle arrest, DNA double-strand break (DSB) repair, genotoxic stress response, and micronuclei formation. Most DEHP metabolizes to mono (2-Ethylhexyl) phthalate (MEHP) and distributes to organs quickly, so MEHP and DEHP were used in cultured cell and animal experiments, respectively. Here, a double blocking mode for the proliferation inhibition of the placental cell was revealed. One is that the classical DSB repair pathways were suppressed, which arrested the cell cycle at the G2/M phase. The other is that DEHP stimulated an elevated level of progesterone, which blocked the cell cycle at metaphase by disrupting chromosome arrangement. These two sets of events facilitated micronuclei formation and resulted in cell proliferation inhibition. This findings provide a novel mechanistic understanding for DEHP to inhibit placental cell proliferation.

Crossover between the adiabatic and nonadiabatic electron transfer limits in the Landau-Zener model.

The semiclassical models of nonadiabatic transition were proposed first by Landau and Zener in 1932, and have been widely used in the study of electron transfer (ET); however, experimental demonstration of the Landau-Zener formula remains challenging to observe. Herein, employing the Hush-Marcus theory, thermal ET in mixed-valence complexes {[Mo2]-(ph)n-[Mo2]}+ (n = 1-3) has been investigated, spanning the nonadiabatic throughout the adiabatic limit, by analysis of the intervalence transition absorbances. Evidently, the Landau-Zener formula is valid in the adiabatic regime in a broader range of conditions than the theoretical limitation known as the narrow avoided-crossing. The intermediate system is identified with an overall transition probability (κel) of ∼0.5, which is contributed by the single and the first multiple passage. This study shows that in the intermediate regime, the ET kinetic results derived from the adiabatic and nonadiabatic formalisms are nearly identical, in accordance with the Landau-Zener model. The obtained insights help to understand and control the ET processes in biological and chemical systems.

Prenatal di-(2-ethylhexyl) phthalate maternal exposure impairs the spatial memory of adult mouse offspring in a phase- and gender-dependent manner.

DEHP is a wildly used plasticizer. Maternal DEHP exposure induced fetal growth restriction (FGR) and behavioral abnormalities in adolescence and adulthood mouse. The effect of low birth weight induced by DEHP on behaviors after growing up is not certain. In this study, the ICR pregnant mice were exposed to 200 mg/kg DEHP during gestation 6-12 days or 13-17 days, which can create FGR model. The F1 offspring were performed three ethological experiments at puberty (6 weeks postpartum) and adult period (8 weeks postpartum). The open field test was performed to detect the locomotor activity and anxiety, the elevated plus maze to test anxiety-like behavior, and the Morris water maze assay to measure the spatial learning and memory capability of male and female offspring. The results showed that spatial memory ability was dramatically impaired for male rather than female offspring in gestation 13-17 days' group. Other behaviors had no statistically different between groups. These findings suggest that prenatal DEHP exposure disturbed mouse offspring spatial memory ability in a phase- and gender-dependent manner.

Lipopolysaccharide downregulates the expressions of intestinal pregnane X receptor and cytochrome P450 3a11.

The pregnane X receptor is a member of the nuclear receptor superfamily, which heterodimerize with the retinoid X receptor, and is an important regulator of cytochrome P450 3A (CYP3A). Lipopolysaccharide (LPS)-induced downregulation of pregnane X receptor and its target gene cyp3a11 has been well characterized in mouse liver. In the present study, we investigated the effects of LPS on the expressions of pregnane X receptor and its target gene cyp3a11 in mouse intestine. Mice were injected intraperitoneally with different doses of LPS (0.1-5.0 mg/kg). Intestinal pregnane X receptor, retinoid X receptor alphalpha and cyp3a11 mRNA were determined using reverse transcription polymerase chain reaction (RT-PCR). Erythromycin N-demethylase (ERND) activity was used as an indicator of CYP3A expression. Results showed that LPS significantly downregulated the expressions of intestinal pregnane X receptor and its heterodimer retinoid X receptor alpha in a dose-dependent manner. Furthermore, LPS repressed the upregulation of cyp3a11 mRNA and ERND catalytic activity in mice pretreated with pregnane X receptor ligand dexamethasone. Additional experiment showed that LPS significantly increased the level of intestinal thiobarbituric acid-reactive substance, which was attenuated by oral administration with either N-acetylcysteine or ascorbic acid. Correspondingly, oral administration with either N-acetylcysteine or ascorbic acid significantly attenuated LPS-induced downregulation of intestinal pregnane X receptor and retinoid X receptor alphalpha. In addition, these antioxidants prevented the repressive effect of LPS on dexamethasone-inducible cyp3a11 mRNA and ERND activity in mouse intestine. Taken together, these results indicate that LPS suppresses the expressions of pregnane X receptor and its target gene cyp3a11 in mouse intestine. LPS-induced downregulation of pregnane X receptor and cyp3a11 in mouse intestine is mediated, at least in part, by oxidative stress.

Melatonin protects against lipopolysaccharide-induced intra-uterine fetal death and growth retardation in mice.

Lipopolysaccharide (LPS) has been associated with adverse developmental outcomes, including intra-uterine fetal death (IUFD) and intra-uterine growth retardation (IUGR). However, the exact mechanism for LPS-induced IUFD and IURD remains unclear. LPS stimulates macrophages to generate reactive oxygen species (ROS). Therefore, we hypothesize that ROS may be involved in LPS-induced IUFD and IURD. Melatonin is a powerful endogenous antioxidant. In this study, we investigated the protective effects of melatonin on LPS-induced IUFD and IURD in ICR mice. All pregnant mice except controls received an intraperitoneal (75 microg/kg, i.p.) injection of LPS on gestational day (gd) 15-17. The experiment was carried out in two different modes. In mode A, the pregnant mice received two doses of melatonin within 24 hr, one (5 or 10 mg/kg) injected immediately after LPS and the other (5 or 10 mg/kg) injected at 3 hr after LPS. In mode B, the pregnant mice were pretreated with 10 mg/kg of melatonin 18 hr before LPS and then received two doses of melatonin in 24 hr, one (10 mg/kg) injected immediately after LPS and the other (10 mg/kg) injected 3 hr after LPS. The number of live fetuses, dead fetuses and resorption sites were counted on gd 18. Live fetuses in each litter were weighed. Crown-rump and tail lengths were examined and skeletal development was evaluated. Results showed that post-treatments with melatonin significantly attenuated LPS-induced IUFD in a dose-dependent manner. Surprisingly, pre- plus post-treatments with melatonin almost blocked LPS-induced IUFD. In addition, both post-treatments and pre- plus post-treatments with melatonin significantly alleviated LPS-induced decreases in crown-rump and tail lengths and reversed LPS-induced skeletal developmental retardation. However, melatonin had little effect on LPS-induced decrease in fetal weight. Furthermore, pre- plus post-treatments with melatonin significantly attenuated LPS-induced lipid peroxidation in maternal liver. These results indicate that melatonin protects against LPS-induced IURD and IUGR via counteracting LPS-induced oxidative stress.

Chronic ethanol exposure downregulates hepatic expression of pregnane X receptor and P450 3A11 in female ICR mice.

Pregnane X receptor (PXR) is a nuclear receptor that regulates cytochrome P450 3A (CYP3A) gene transcription in a ligand-dependent manner. Ethanol has been reported to be either an inducer or an inhibitor of CYP3A expression. In this study, we investigated the effects of chronic ethanol exposure on PXR and P450 3A11 gene expression in mouse liver. Female ICR mice were administered by gavage with different doses (1000, 2000 and 4000 mg/kg) of ethanol for up to 5 weeks. Hepatic PXR and P450 3A11 mRNA levels were measured using RT-PCR. Erythromycin N-demethylase (ERND) activity was used as an indicator of CYP3A protein expression. Results showed that chronic ethanol exposure markedly decreased hepatic PXR and P450 3A11 mRNA levels. Consistent with downregulation of P450 3A11 mRNA, chronic ethanol exposure significantly decreased ERND activity in a dose-dependent manner. Additional experiment showed that chronic ethanol exposure significantly increased plasma endotoxin level and hepatic CD14 and TLR-4 mRNA expression, all of which were blocked by elimination of Gram-negative bacteria and endotoxin with antibiotics. Correspondingly, pretreatment with antibiotics reversed the downregulation of PXR and P450 3A11 mRNA expression and ERND activity in mouse liver. Furthermore, the downregulation of hepatic PXR and P450 3A11 mRNA expression was significantly attenuated in mice pretreated with GdCl(3), a selective Kupffer cell toxicant. GdCl(3) pretreatment also significantly attenuated chronically ethanol-induced decrease in ERND activity. These results indicated that activation of Kupffer cells by gut-derived endotoxin contributes to downregulation of hepatic PXR and P450 3A11 expression during chronic alcohol intoxication.

Perinatal lipopolysaccharide exposure downregulates pregnane X receptor and Cyp3a11 expression in fetal mouse liver.

The pregnane X receptor (PXR) is a member of the nuclear receptor superfamily that regulates cytochrome P450 3A (CYP3A) gene transcription in a ligand-dependent manner. Lipopolysaccharide (LPS)-induced downregulation on PXR and cyp3a11 in adult mouse liver has been well characterized. In this study, we investigated the effects of maternal LPS exposure on PXR and cyp3a11 expression in fetal mouse liver. Pregnant ICR mice were injected intraperitoneally with different doses of LPS (0.1 approximately 0.5 mg/kg) on gestational day (GD) 17. PXR and cyp3a11 mRNA levels were determined using RT-PCR. Erythromycin N-demethylase (ERND) activity was used as an indicator of CYP3A expression in this study. Results showed that LPS significantly downregulated PXR and cyp3a11 mRNA levels and ERND activity in fetal liver in a dose-dependent manner. LPS-induced downregulation of PXR and cyp3a11 mRNA expression and ERND activity was attenuated after pregnant mice were pretreated with alpha-phenyl-N-t-butylnitrone (PBN), a free radical spin trapping agent. Additional experiment revealed that LPS significantly increased lipid peroxidation in fetal liver, which was also attenuated by PBN pretreatment. Furthermore, LPS-induced downregulation of PXR and cyp3a11 mRNA expression and ERND activity was prevented by maternal pretreatment with N-acetylcysteine (NAC). Maternal pretreatment with NAC also inhibited LPS-initiated lipid peroxidation and GSH depletion in fetal liver. However, maternal LPS treatment did not affect nitrite plus nitrate concentration in fetal liver. Correspondingly, aminoguanidine, a selective inhibitor of inducible nitric oxide synthase (iNOS), has no effect on LPS-induced downregulation of PXR and cyp3a11 expression and ERND activity in fetal liver. These results indicated that maternal LPS exposure downregulates PXR and cyp3a11 in fetal mouse liver. Reactive oxygen species (ROS) may be involved in LPS-induced downregulation of PXR and cyp3a11 in fetal mouse liver.

Lipopolysaccharide treatment downregulates the expression of the pregnane X receptor, cyp3a11 and mdr1a genes in mouse placenta.

The cytochrome P450 3A (CYP3A) is a member of the cytochrome P450 monooxygenase superfamily. The multidrug resistance 1 (MDR1) gene belongs to the ATP-binding cassette (ABC) family. Pregnane X receptor (PXR) is a nuclear receptor that regulates its target gene transcription in a ligand-dependent manner. Lipopolysaccharide (LPS)-induced downregulation of PXR, CYP3A and MDR1 in liver has been demonstrated in a series of studies. However, it is not clear whether LPS represses the expression of PXR, CYP3A and MDR1 in placenta. In the present study, we investigated the effects of LPS on the expression of PXR, cyp3a11 and mdr1a in mouse placenta. Pregnant ICR mice were injected intraperitoneally with different doses of LPS (0.1-0.5 mg/kg) on gestational day (gd) 17. Placental PXR, cyp3a11 and mdr1a mRNA levels were determined at 12 h after LPS treatment using RT-PCR. Results showed that LPS significantly downregulated PXR, cyp3a11 and mdr1a mRNA levels in a dose-dependent manner. LPS-induced downregulation of PXR, cyp3a11 and mdr1a mRNA in placenta was significantly attenuated after pregnant mice were pre- and post-treated with alpha-phenyl-N-t-butylnitrone (PBN), a free radical spin trapping agent. Additional experiments revealed that LPS increased lipid peroxidation and proinflammatory cytokine expressions in mouse placenta, all of which were also attenuated by PBN. Furthermore, LPS-induced downregulation of PXR, cyp3a11 and mdr1a mRNA in mouse placenta was prevented by N-acetylcysteine (NAC). NAC also inhibited LPS-initiated lipid peroxidation, GSH depletion and proinflammatory cytokine expressions in mouse placenta. These results indicated that LPS downregulates placental PXR, cyp3a11 and mdr1a mRNA expressions. Reactive oxygen species (ROS) may be involved in LPS-induced downregulation of PXR, cyp3a11 and mdr1a in mouse placenta.

Melatonin attenuates lipopolysaccharide-induced down-regulation of pregnane X receptor and its target gene CYP3A in mouse liver.

Pregnane X receptor (PXR) is a member of the nuclear receptor superfamily that regulates target gene transcription in a ligand-dependent manner. Our earlier study indicated that reactive oxygen species contribute to lipopolysaccharide (LPS)-induced down-regulation of PXR and its target gene CYP3A in mouse liver. Melatonin is a powerful endogenous antioxidants. In this study, we investigated the effects of melatonin on LPS-induced down-regulation of PXR and CYP3A in mouse liver. Mice were intraperitoneally administrated different doses of melatonin before and/or after LPS treatment. PXR and CYP3A11 mRNA levels were measured using RT-PCR. Erythromycin N-demethylase (ERND) was used as an indicator of CYP3A catalytic activity. Results indicated that melatonin significantly attenuated LPS-induced down-regulation of PXR and CYP3A11 mRNA levels in a dose-dependent manner. Repeated doses of melatonin (10 mg/kg) treatments also significantly attenuated LPS-induced down-regulation of dexamethasone-inducible CYP3A11 mRNA level and ERND activity in mouse liver. In addition, the present study also shows that melatonin significantly increased hepatic superoxide dismutase, Se-dependent glutathione peroxidase, glutathione reductase and catalase activities and glutathione levels in LPS-treated mice. These findings suggest that melatonin may exert its protective effects on LPS-induced down-regulation of PXR and CYP3A via counteracting LPS-induced oxidative stress in mouse liver.

Kupffer cells and reactive oxygen species partially mediate lipopolysaccharide-induced downregulation of nuclear receptor pregnane x receptor and its target gene CYP3a in mouse liver.

Pregnane X receptor (PXR) is a member of the nuclear receptor superfamily that regulates target gene transcription in a ligand-dependent manner. The in vivo effects of lipopolysaccharide (LPS) on expression of PXR and its target gene cytochrome P450 3A (CYP3A) in mouse liver were investigated in this study. Mice were injected intraperitoneally with different doses of LPS (0.1-5.0 mg/kg). PXR and CYP3A11 mRNA levels were measured using reverse transcription polymerase chain reaction. Results indicate that LPS significantly inhibits the expression of PXR mRNA in a dose-dependent manner, followed by suppression of CYP3A11 mRNA in mouse liver. LPS also represses the upregulation of CYP3A11 mRNA levels and erythromycin N-demethylase (ERND) catalytic activity in mice pretreated with PXR ligands dexamethasone, rifampicin, mifepristone, and phenobarbital. LPS-induced downregulation of PXR and CYP3A11 mRNA in liver was significantly attenuated in mice pretreated with gadolinium chloride, a selective Kupffer cell toxicant. Pretreatment with a single dose of gadolinium chloride (10 mg/kg) also significantly attenuated LPS-induced downregulation of dexamethasone-, rifampicim-, mifepristone-, and phenobarbital-inducible, CYP3A11 mRNA expression and ERND activity in mouse liver. Furthermore, LPS-induced downregulation of PXR and CYP3A11 mRNA was significantly attenuated in mice pretreated with allopurinol, an inhibitor of xanthine oxidase, and diphenyleneiodonium chloride, an inhibitor of NADPH oxidase. Allopurinol and diphenyleneiodonium chloride pretreatment also attenuated the repressive effects of LPS on dexamethasone-, rifampicin-, mifepristone-, and phenobarbital-inducible CYP3A11 mRNA expression and ERND catalytic activity in mouse liver. However, aminoguanidine, a selective inhibitor of inducible nitric oxide synthase, has no effect on LPS-induced downregulation of PXR and CYP3A11 mRNA. Finally, LPS-induced downregulation of PXR and CYP3A11 mRNA was prevented in mice pretreated with either N-acetylcysteine or ascorbic acid. These antioxidants also prevented the repressive effects of LPS on dexamethasone-, rifampicin-, mifepristone-, and phenobarbital-inducible CYP3A11 mRNA expression and ERND catalytic activity in mouse liver. These results indicate that Kupffer cells contribute to LPS-induced downregulation of PXR and CYP3A in mouse liver. Reactive oxygen species, produced possibly by NADPH oxidase and perhaps by xanthine oxidase, are involved in LPS-induced downregulation of nuclear receptor PXR and its target gene CYP3A in mouse liver.