Cytochrome P450 3A1 Mediates 2,2′,4,4′-Tetrabromodiphenyl Ether-Induced Reduction of Spermatogenesis in Adult Rats

Background

2,2′,4,4′-tetrabromodiphenyl ether (BDE47) is the dominant PBDE congener in humans, wildlife, and the environment. It has been reported to be metabolized by cytochrome P450 (CYP) enzymes. Still, the effects of BDE47 on spermatogenesis failure are attracting an increasing amount of attention. However, it is unclear whether CYP-mediated metabolism contributes to BDE47-induced reproductive toxicity.

Methodology and Principal Findings

The role of cytochrome P450 3A1 (CYP3A1) in the formation of oxidative metabolites of BDE47 and its induced spermatogenesis failure was investigated in SD rats. BDE47 significantly increased the expression and activity of CYP3A1 in rat liver, and 3-OH-BDE47, the major oxidative metabolite of BDE47, dose-dependently increased in rat liver, serum, and testis, which was aggravated by dexamethasone (DEX), an inducer of CYP3A1. Additionally, testicular 3-OH-BDE47 and reactive oxygen species (ROS) in seminiferous tubules increased especially when BDE47 was administered in combination with DEX, which was confirmed in GC-1 and GC-2 cells that 3-OH-BDE47 induced more ROS production and cell apoptosis via the upregulation of FAS/FASL, p-p53 and caspase 3. As a result, daily sperm production dose-dependently decreased, consistent with histological observations in giant cells and vacuolar spaces and increase in TUNEL-positive apoptotic germ cells.

Conclusion

CYP3A1-mediated metabolic activation of BDE47 and the active metabolite 3-OH-BDE47 and consequent ROS played an important role in reduction of spermatogenesis by germ cell apoptosis. Our study helps provide new insights into the mechanism of reproductive toxicity of environmental chemicals.     

Isolation of a 1 195 bp 5′-Flanking Region of Rice Cytosolic Fructose-1,6-bisphosphatase and Analysis of Its Expression in Transgenic Rice

A genomic DNA fragment containing the 5′-upstream sequence and part of the open reading frame corresponding to the cytosolic fructose-1,6-bisphosphatase (cyFBPase) cDNA was isolated by Genome Walking. The 1 195 bp 5′-flanking region which started from the translation initiation ATG codon was fused to reporter gene encoding β-glucuronidase (GUS) and stably transferred to rice via particle bombardment. Strong GUS activity was detected in leaves and leaf sheaths of transgenic rice, but not in culms and roots. Histochemical localization revealed that GUS expression was exclusively restricted to mesophyll cells in transgenic rice. Our results indicate that the 1 195 bp fragment contains all the cis-elements required for directing mesophyll-specific expression pattern in rice. Key words: rice (Oryza sativa); promoter;　cytosolic fructose-1,6-bisphosphatase gene; mesophyll-specific expression     

Polymorphism of the 5′ Untranslated Region of NHE1 Gene Associated with Type-I Diabetes

The ubiquitous form of the sodium–hydrogen exchanger, NHE1, is devoted to the regulation of intracellular pH and cell volume. In addition, NHE1 activity is stimulated by growth factors and increased NHE rates are found in both circulating and immortalized cells during diabetes or diabetic nephropathy. In this context, we searched for polymorphisms of the 5′-flanking regulatory region of NHE1 gene in subjects with type-I diabetes. We identified a C/T transition 696 bases upstream the translation initiation start site which disrupts a repeated palindromic GC sequence. The TT genotype was significantly more frequent in type-1 diabetics and may have functional importance. Genetic linkage between NHE1 and diabetes has been previously described in NOD mice strains with consequences on NHE rates. Hence, the polymorphism described hereby may act as a predisposition factor to type-I diabetes or to diabetic complications, and may be useful to investigate the genetic involvement of NHE1 in human pathophysiology.     

Cytochrome P450c17α 5′-untranslated region *T/C polymorphism in endometriosis

Estrogen plays a role in the pathogenesis of endometriosis. The CYP17 gene codes for the cytochrome P450c17α enzyme that is involved in the estrogen biosynthesis. We aimed to investigate if CYP17 polymorphism could be used as marker to predict the susceptibility of endometriosis. Women were divided into two groups: (1) severe endometriosis (n=119); (2) non-endometriosis groups (n=128). A 169-bp fragment encompassing the T/C polymorphic site in 5′-untranslated promoter region (5′-UTR) of the CYP17 was amplified by the polymerase chain reaction, treated with restriction enzyme MspA1I, and electrophoresis. The polymorphism was divided into restriction-enzyme indigestible (T homozygote), T/C heterozygote, and digestible (C homozygote). Genotypes and allelic frequencies for this polymorphism in both groups were compared. We observed a higher but non-significant percentage of T homozygote in the endometriosis women compared with the non-endometriosis women. Proportions of T homozygote/heterozygote/C homozygote for CYP17 in both groups were: (1) 26.1/46.2/27.7% and (2) 17.2/45.3/37.5% (p-value=0.131). T allele was related with higher susceptibility of endometriosis. T and C allele frequencies in both groups were: (1) 49.2/50.8%; (2) 39.8/60.2% (p-value=0.046). Despite the CYP17* T allele appearing to be asscoiatd with a trend of increased risk of endometriosis, CYP17 5′-UTR gene polymorphism might not be a useful marker for prediction of endometriosis susceptibility.     

An EAV-HP Insertion in 5′ Flanking Region of SLCO1B3 Causes Blue Eggshell in the Chicken

The genetic determination of eggshell coloration has not been determined in birds. Here we report that the blue eggshell is caused by an EAV-HP insertion that promotes the expression of SLCO1B3 gene in the uterus (shell gland) of the oviduct in chicken. In this study, the genetic map location of the blue eggshell gene was refined by linkage analysis in an F₂ chicken population, and four candidate genes within the refined interval were subsequently tested for their expression levels in the shell gland of the uterus from blue-shelled and non-blue-shelled hens. SLCO1B3 gene was found to be the only one expressed in the uterus of blue-shelled hens but not in that of non-blue-shelled hens. Results from a pyrosequencing analysis showed that only the allele of SLCO1B3 from blue-shelled chickens was expressed in the uterus of heterozygous hens (O*LC/O*N). SLCO1B3 gene belongs to the organic anion transporting polypeptide (OATP) family; and the OATPs, functioning as membrane transporters, have been reported for the transportation of amphipathic organic compounds, including bile salt in mammals. We subsequently resequenced the whole genomic region of SLCO1B3 and discovered an EAV-HP insertion in the 5′ flanking region of SLCO1B3. The EAV-HP insertion was found closely associated with blue eggshell phenotype following complete Mendelian segregation. In situ hybridization also demonstrated that the blue eggshell is associated with ectopic expression of SLCO1B3 in shell glands of uterus. Our finding strongly suggests that the EAV-HP insertion is the causative mutation for the blue eggshell phenotype. The insertion was also found in another Chinese blue-shelled breed and an American blue-shelled breed. In addition, we found that the insertion site in the blue-shelled chickens from Araucana is different from that in Chinese breeds, which implied independent integration events in the blue-shelled chickens from the two continents, providing a parallel evolutionary example at the molecular level.     

Cytochrome P450 2E1 potentiates ethanol induction of hypoxia and HIF-1α in vivo

Ethanol induces hypoxia and elevates HIF-1α in the liver. CYP2E1 plays a role in the mechanisms by which ethanol generates oxidative stress, fatty liver, and liver injury. This study evaluated whether CYP2E1 contributes to ethanol-induced hypoxia and activation of HIF-1α in vivo and whether HIF-1α protects against or promotes CYP2E1-dependent toxicity in vitro. Wild-type (WT), CYP2E1-knock-in (KI), and CYP2E1 knockout (KO) mice were fed ethanol chronically; pair-fed controls received isocaloric dextrose. Ethanol produced liver injury in the KI mice to a much greater extent than in the WT and KO mice. Protein levels of HIF-1α and downstream targets of HIF-1α activation were elevated in the ethanol-fed KI mice compared to the WT and KO mice. Levels of HIF prolyl hydroxylase 2, which promotes HIF-1α degradation, were decreased in the ethanol-fed KI mice in association with the increases in HIF-1α. Hypoxia occurred in the ethanol-fed CYP2E1 KI mice as shown by an increased area of staining using the hypoxia-specific marker pimonidazole. Hypoxia was lower in the ethanol-fed WT mice and lowest in the ethanol-fed KO mice and all the dextrose-fed mice. In situ double staining showed that pimonidazole and CYP2E1 were colocalized to the same area of injury in the hepatic centrilobule. Increased protein levels of HIF-1α were also found after acute ethanol treatment of KI mice. Treatment of HepG2 E47 cells, which express CYP2E1, with ethanol plus arachidonic acid (AA) or ethanol plus buthionine sulfoximine (BSO), which depletes glutathione, caused loss of cell viability to a greater extent than in HepG2 C34 cells, which do not express CYP2E1. These treatments elevated protein levels of HIF-1α to a greater extent in E47 cells than in C34 cells. 2-Methoxyestradiol, an inhibitor of HIF-1α, blunted the toxic effects of ethanol plus AA and ethanol plus BSO in the E47 cells in association with inhibition of HIF-1α. The HIF-1α inhibitor also blocked the elevated oxidative stress produced by ethanol/AA or ethanol/BSO in the E47 cells. These results suggest that CYP2E1 plays a role in ethanol-induced hypoxia, oxidative stress, and activation of HIF-1α and that HIF-1α contributes to CYP2E1-dependent ethanol-induced toxicity. Blocking HIF-1α activation and actions may have therapeutic implications for protection against ethanol/CYP2E1-induced oxidative stress, steatosis, and liver injury.     

Involvement of Cytochrome P450 1A1 and Glutathione S-Transferase P1 Polymorphisms and Promoter Hypermethylation in the Progression of Anti-Tuberculosis Drug-Induced Liver Injury: A Case–Control Study

Background

Anti-tuberculosis (anti-TB) drug-induced liver injury (ADLI) is one of the most common adverse effects associated with TB treatment. Cytochrome P450 (CYP) 1A1 and glutathione S-transferase (GST) P1 are important phase I/II metabolizing enzymes involved in drug metabolism and detoxification. Genetic polymorphism and CpG island methylation have been reported as factors influencing the expression of CYP1A1 and GSTP1.

Objective

This study aimed to determine the potential relationships of CYP1A1 and GSTP1 polymorphisms and CpG island methylation with ADLI risk.

Design

This was a population-based one-to-one matched case–control study.

Setting

The subjects were patients with TB receiving treatment in China from December 2010 to June 2013.

Patients

In total, 127 patients with TB and ADLI (case group) and 127 patients with TB but without liver injury (control group) were included in this study. Subjects were matched in terms of sex, age, and therapeutic regimen.

Methods

The general condition of each patient was assessed using questionnaires. The CYP1A1 MspI and GSTP1 Ile105Val polymorphisms as well as methylation status were detected by polymerase chain reaction (PCR)–restriction fragment length polymorphism and the methylation-specific PCR method.

Results

We found no significant difference in GSTP1 and CYP1A1 genotypes between the two groups, probably because the sample size was not large enough; however, patients with ADLI had significantly higher GSTP1 and CYP1A1 promoter methylation rates than control subjects [odds ratio (OR) = 2.467 and 2.000, respectively]. After adjusting for drinking, which significantly differed between the groups as per univariate analysis, we found that hypermethylation of GSTP1 and CYP1A1 promoters was associated with ADLI (OR = 2.645 and 2.090, respectively).

Conclusion

Hypermethylation of CpG islands of GSTP1 and CYP1A1 promoters may thus play important roles in the development of ADLI and provide evidence of being used as novel markers for ADLI risk prediction.