Disruption of thyroid hormone homeostasis in Ugt1a-deficient Gunn rats by microsomal enzyme inducers is not due to enhanced thyroxine glucuronidation

Microsomal enzyme inducers (MEI) that increase UDP-glucuronosyltransferases (UGTs) are thought to increase glucuronidation of thyroxine (T₄), thus reducing serum T₄, and subsequently increasing thyroid stimulating hormone (TSH). Ugt1a1 and Ugt1a6 mediate T₄ glucuronidation. Therefore, this experiment determined the involvement of Ugt1a enzymes in increased T₄ glucuronidation, decreased serum T₄, and increased TSH after MEI treatment. Male Wistar and Ugt1a-deficient Wistar (Gunn) rats were fed a control diet or diet containing pregnenolone-16α-carbonitrile (PCN; 800 ppm), 3-methylcholanthrene (3-MC; 200 ppm), or Aroclor 1254 (PCB; 100 ppm) for 7 days. Serum T₄, triiodothyronine (T₃), and TSH concentrations, hepatic T₄/T₃ glucuronidation, and thyroid histology and follicular cell proliferation were investigated. PCN, 3-MC, and PCB treatments decreased serum T₄, whereas serum T₃ was maintained in both Gunn and Wistar rats (except for PCB treatment). TSH was increased in Wistar and Gunn rats after PCN (130 and 277%) or PCB treatment (72 and 60%). T₄ glucuronidation in Wistar rats was increased after PCN (298%), 3-MC (85%), and PCB (450%), but was extremely low in Gunn rats, and unchanged after MEI. T₃ glucuronidation was increased after PCN (121%) or PCB (58%) in Wistar rats, but only PCN increased T₃ glucuronidation in Gunn rats (43%). PCN treatment induced thyroid morphological changes and increased follicular cell proliferation in both strains. These data demonstrate that T₄ glucuronidation cannot be increased in Ugt1a-deficient Gunn rats. Thus, the decrease in serum T₄, increase in TSH, and increase in thyroid cell proliferation after MEI are not dependent on increased T₄ glucuronidation, and cannot be attributed to Ugt1a enzymes.     

Genetic polymorphisms of UGT1A6 in a Japanese population

Thirteen single nucleotide polymorphisms (SNPs), including 6 novel ones, were found in exon 1 and its flanking region of UDP-glucuronosyltransferase (UGT) 1A6 from 195 Japanese subjects. Several novel SNPs were identified, including 269G>A (R90H), 279A>G (S93S), and 308C>A (S103X) in exon 1, and IVS1+109C>T, IVS1+120A>G, and IVS1+142C>T in the intron downstream of exon 1. Among these SNPs, 308C>A confers termination of translation at codon 103, resulting in the production of an immature protein that probably lacks enzymatic activity. The allele frequencies were 0.003 for all the 6 SNPs. In addition, the 3 known nonsynonymous SNPs were detected: 19T>G (S7A), 541A>G (T181A), and 552A>C (R184S) with frequencies of 0.226, 0.218, and 0.226, respectively. High linkage disequilibrium was observed among 19T>G (S7A), 315A>G (L105L), 541A>G (T181A), 552A>C (R184S), and IVS1+130G>T, as reported in Caucasian and African-American populations. Then, 11 haplotypes in UGT1A6 were estimated. The novel nonsynonymous variant, 269A or 308A, was shown to be located on the same DNA strand together with 19G, 315G, 541G, 552C, and IVS1+130T. Our results provide fundamental and useful information for genotyping UGT1A6 in the Japanese, and probably Asian populations.     

Effect of a conserved mutation in uridine diphosphate glucuronosyltransferase 1A1 and 1A6 on glucuronidation of a metabolite of flutamide

OBJECTIVE: We investigated how the conserved mutation (Y486D) changed the kinetic parameters of uridine diphosphate glucuronosyltransferase 1A1 and 1A6 (UGT1A1 and 1A6) for 2-amino-5-nitro-4-trifluoromethylphenol, which is a major metabolite of flutamide, a nonsteroidal antiandrogenic agent. METHODS: The wild-type or mutant cDNA-expressed UGT was co-incubated with 2-amino-5-nitro-4-trifluoromethylphenol (aglycone) and uridine diphosphate-glucuronic acid (UDP-GA, donor substrate). The glucuronidation of the aglycone was determined. RESULTS: The maximum velocities of the mutant UGT1A1 and UGT1A6 were about 12% and less than 1% of the corresponding wild-type, respectively. The Michaelis constant (K(M)) for the aglycone of the wild-type UGT1A1 was double that of the mutant, but the K(M) for UDP-GA of the wild-type UGT1A1 was not significantly different from that of the mutant. CONCLUSION: Patients with Y486D may accumulate excessive 2-amino-5-nitro-4-trifluoromethylphenol, which might lead to unexpected toxicity.     

1-Aryl-6-azauracils,XXXVI1): Synthesis of 6-Azauracil Derivatives of Phenazone

In spite of the fact that a great number of several 4-substituted derivatives of phenazone has already been prepared, the only derivative with 6-azauracile cycle up to now is 4-(2-thio-6azauracil-5-yl)-phenazone, prepared by Schmidt²⁾.     

6-氨基-1-丙基-尿嘧啶的合成

6-氨基-1-丙基-尿嘧啶是合成嘌呤及嘌呤衍生物的医药中间体,目前为止国内没有生产的,经多次试验确定以丙基脲,氰乙酸,酸酐为原料,通过亲核反应,再在氢氧化钠的作用下合环生成6-氨基-1-丙基-尿嘧啶,此合成总收率〉70%。     

Mercury modulates the cytochrome P450 1a1, 1a2 and 1b1 in C57BL/6J mice: in vivo and in vitro studies

In the current study C57BL/6J mice were injected intraperitoneally with Hg^2 + in the absence and presence of TCDD. After 6 and 24 h the liver was harvested and the expression of Cyps was determined. In vitro, isolated hepatocytes were incubated with TCDD in the presence and absence of Hg^2 +. At the in vivo level, Hg^2 + significantly decreased the TCDD-mediated induction of Cyps at 6 h while potentiating their levels at 24 h. In vitro, Hg^2 + significantly inhibited the TCDD-mediated induction of Cyp1a1 in a concentration- and time-dependent manner. Interestingly, Hg^2 + increased the serum hemoglobin (Hb) levels in mice treated for 24 h. Upon treatment of isolated hepatocytes with Hb alone, there was an increase in the AhR-dependent luciferase activity with a subsequent increase in Cyp1a1 protein and catalytic activity levels. Importantly, when hepatocytes were treated for 2 h with Hg^2 + in the presence of TCDD, then the medium was replaced with new medium containing Hb, there was potentiation of the TCDD-mediated effect. In addition, Hg^2 + increased heme oxygenase-1 (HO-1) mRNA, which coincided with a decrease in the Cyp1a1 activity level. When the competitive HO-1 inhibitor, tin mesoporphyrin was applied to the hepatocytes there was a partial restoration of Hg^2 +-mediated inhibition of Cyp1a1 activity. In conclusion, we demonstrate for the first time that there is a differential modulation of the TCDD-mediated induction of Cyp1a1 by Hg^2 + in C57BL/6J mice livers and isolated hepatocytes. Moreover, this study implicates Hb as an in vivo specific modulator of Cyp1 family.     

Structural characterization of a new variant of the CYP2A6 gene (CYP2A6*1B) apparently diagnosed as heterozygotes of CYP2A6*1A and CYP2A6*4C

During the course of investigating the frequency of a CYP2A6 whole deletion-type polymorphism (CYP2A6*4C) in Japanese, an unexpectedly large population of heterozygotes for CYP2A6*4C and the wild-type (CYP2A6*1A) was found. Cloning of a cDNA encoding CYP2A6 from the liver of individuals judged as heterozygotes for CYP2A6*4C and the CYP2A6*1A was carried out to identify the causal allele(s) responsible for a possible overestimation. A clone isolated from the liver cDNA library possessed 58 bp sequences in the 3'-untranslated region, which was replaced with the corresponding region of the CYP2A7 gene. The same gene conversion existed in the genomic DNA, indicating that the replacement was not a cloning artifact. Based on the gene structure of the allele (CYP2A6*1B), this variant was thought to be one of the causal alleles responsible for overestimation of heterozygotes for CYP2A6*4C and CYP2A6* A. To investigate this further, we developed a genotyping method which could distinguish the CYP2A6*A, CYP2A6*1B and CYP2A6*4C alleles from each other. The results clearly showed that CYP2A6*1B was the sole allele responsible for the overestimation. We conclude that the new genotyping method allows determination of six genotypes of the CYP2A6 gene, simultaneously and precisely, in both Oriental and Caucasian populations.     

CYP2D6 and GSTM1 genotypes in a Polish population

Objective: The aim of the present study was to investigate the distribution of the CYP2D6 and GSTM1 genotypes in a Polish population. Methods: One hundred and forty-five unrelated healthy individuals from the western region of Poland were studied. The CYP2D6 genotype was analysed by means of polymerase chain reaction (PCR) amplification for the CYP2D6 ^* 3 and CYP2D6 ^* 4 alleles. The GSTM1 genotype was also analysed by means of a PCR assay to determine two genotypes: GSTM1-1 (positive) and GSTM1-0 (negative). Results: Fourteen subjects (9.6%) were classified as poor metabolisers. The frequency of CYP2D6 ^* 4 and CYP2D6 ^* 3 was 23.1% and 2.1%, respectively. The frequency of GSTM1 nulled genotype in a Polish population came to 49%. Conclusion: The frequencies of poor metabolisers for CYP2D6 and GSTM1 nulled genotype among a Polish population were similar to those observed in other Caucasian populations. Received: 29 October 1998 / Accepted in revised form: 15 March 1999     

Pharmacological properties of pempidine (1:2:2:6:6-pentamethylpiperidine), a new ganglion-blocking compound

Pempidine (1:2:2:6:6-pentamethylpiperidine) is a long-acting ganglion-blocking compound which is effective by mouth. By intravenous injection it has a similar potency to hexamethonium on the preganglionically stimulated nictitating membrane of the cat. The compound blocks the effects of intravenous nicotine and of peripheral vagal stimulation on the blood pressure; it also causes dilatation of the pupil after removal of the sympathetic innervation. On the guinea-pig ileum, the predominant effect of the compound is to inhibit nicotine contractions. Pempidine is well absorbed from the gastro-intestinal tract as judged by (a) the low ratio (6.9) of oral to intravenous toxicities, (b) the rapid development of mydriasis in mice after oral administration of small doses, and (c) the rapid onset of hypotension when the compound is injected directly into the duodenum of anaesthetized cats. Other actions include neuromuscular paralysis of curare-like type when large doses of the compound are injected intravenously and central effects such as tremors which occur with near toxic doses. In cats with a low blood pressure, large intravenous doses have a slight pressor action.     

A potent PGK1 antagonist reveals PGK1 regulates the production of IL-1β and IL-6

Glycolytic metabolism enzymes have been implicated in the immunometabolism field through changes in metabolic status. PGK1 is a catalytic enzyme in the glycolytic pathway. Here, we set up a high-throughput screen platform to identify PGK1 inhibitors. DC-PGKI is an ATP-competitive inhibitor of PGK1 with an affinity of K_d = 99.08 nmol/L. DC-PGKI stabilizes PGK1 in vitro and in vivo, and suppresses both glycolytic activity and the kinase function of PGK1. In addition, DC-PGKI unveils that PGK1 regulates production of IL-1β and IL-6 in LPS-stimulated macrophages. Mechanistically, inhibition of PGK1 with DC-PGKI results in NRF2 (nuclear factor-erythroid factor 2-related factor 2, NFE2L2) accumulation, then NRF2 translocates to the nucleus and binds to the proximity region of Il-1β and Il-6 genes, and inhibits LPS-induced expression of these genes. DC-PGKI ameliorates colitis in the dextran sulfate sodium (DSS)-induced colitis mouse model. These data support PGK1 as a regulator of macrophages and suggest potential utility of PGK1 inhibitors in the treatment of inflammatory bowel disease.     

6-Keto-prostaglandin E1 inhibits the aggregation of human platelets.

6-Keto-prostaglandin E1 (6-keto-PGE1) was found to have a similar potency to prostacyclin (PGI2) as an inhibitor of platelet aggregation. It caused a time and concentration dependent inhibition of ADP, collagen and epinephrine induced platelet aggregation, the dose ratios for 70% inhibition by 6-keto-PGE1, PGI2 and PGE1 being approximately 1 : 1 : 13. In doses similar to those of PGI2, 6-keto-PGE1 partially inhibited the release of [3H]-serotonin from platelet-rich plasma induced by collagen.     

Fructose 1, 6 bisphosphatase activities in mouse liver as a function of age

Neutral fructose 1, 6 bisphosphatase activity increases till 7 days, after which, a decline is observed postnatally upto 30 days. Alkaline fructose 1, 6 bisphosphatase follows the same pattern. The optimum activity of fructose 1, 6 bisphosphatase in mouse liver at pH 6.5 and 9.0 of all the periods, suggests the presence of both neutral and alkaline enzyme during the developmental period studied. On the basis of similarity observed in optimum pH, the same properties of enzyme at all the developmental stages studied, could not be ruled out.     

Genetic polymorphisms in CYP1A1, CYP2D6, UGT1A6, UGT1A7, and SULT1A1 genes and correlation with benzene exposure in a Chinese occupational population

Metabolic enzymes involved in benzene activation or detoxification, including cytochrome P-450 1A1 (CYP1A1), cytochrome P-450 2D6 (CYP2D6), UDP-glucuronosyltransferase 1A6 (UGT1A6), UDP-glucuronosyltransferase1A7 (UGT1A7), and sulfotransferase 1A1 (SULT1A1), were studied for their roles in human susceptibility to benzene poisoning. All 304 subjects were investigated with a unitary questionnaire and their DNA was isolated from blood samples by a routine phenol-chloroform extraction. The study included 152 benzene poisoning patients, and 152 control workers occupationally exposed to benzene in South China. The genotypes were determined by polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP) technique with genomic DNA. No individuals had the CYP 2D6 c.212 G>A variant alleles in this study. There is no association between the UGT1A6 c.181 T>A, UGT1A7 c.208 Trp>Arg, and SULT1A1 c.638 G>A genotypes and increased risk of benzene-induced carcinogenesis. Although most of the CYP2D6 haplotypes did not show any significant difference, the CYP2D6 haplotype CYP2D6 c.188 C/C, C/T, and c.4268 C/C was significantly overrepresented in the case group (OR 4.02, 95% CI: 2.53-6.39) compared with in controls. Overall, our data suggested that individuals with CYP1A1 c.5639 T/T, CYP2D6 c.188 C/C, C/T, and CYP2D6 c.4268 C/C genotypes tend to be more susceptible to benzene toxicity.     

Hypotensive and renovascular actions of 6-keto-prostaglandin E1, a metabolite of prostacyclin.

Either intra-aortic or intravenous injections of a stable prostaglandin metabolite, 6-keto-prostaglandin E1 (6-keto-PGE1), caused similar dose-dependent falls in blood pressure and reductions in renovascular resistance in the anesthetized rat. The threshold dose was 0.3 microgram/kg. A maximum hypotensive effect occured at 10 micrograms/kg, but renal blood flow was further reduced by a dose of 30 microgram/kg. 6-keto-PGE1, like prostacyclin, could be a circulating hormone.