Human carcinogenic risk evaluation, Part V: The national toxicology program vision for assessing the human carcinogenic hazard of chemicals.

The National Toxicology Program (NTP) has over 25 years of experience in the design, performance, and interpretation of assays for identifying carcinogenic hazards to humans. Through the years we have examined alternative assays and adjunct assays to the standard rodent cancer bioassay including batteries of genetic toxicity tests and genetically modified mouse models. As our collective understanding of carcinogenesis advances, toxicologists and regulatory scientists will at some point begin to rely on mechanism-based biological observations rather than the two-year rodent bioassay to predict human cancer hazards. The goal of the NTP Vision for the 21st Century is to develop the science base that will advance the use of mechanism-based biological observations, eventually providing a replacement for disease-specific toxicology models in the protection of public health.     

Induction of human cytochrome P450 3A4 by the irreversible myeloperoxidase inactivator PF-06282999 is mediated by the pregnane X receptor

1. 2-(6-(5-Chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl) acetamide (PF-06282999) is a member of the thiouracil class of irreversible inactivators of human myeloperoxidase enzyme and a candidate for the treatment of cardiovascular disease. PF-06282999 is an inducer of CYP3A4 mRNA and midazolam-1′-hydroxylase activity in human hepatocytes, which is consistent with PF-06282999-dose dependent decreases in mean maximal plasma concentrations (C_max) and area under the plasma concentration time curve (AUC) of midazolam in humans following 14-day treatment with PF-06282999.

2. In the present study, the biochemical mechanism(s) of CYP3A4 induction by PF-06282999 was studied. Incubations in reporter cells indicated that PF-06282999 selectively activated human pregnane X receptor (PXR). Treatment of human HepaRG cells with PF-06282999 led to ～14-fold induction in CYP3A4 mRNA and 5-fold increase in midazolam-1′-hydroxylase activity, which was nullified in PXR-knock out HepaRG cells. TaqMan® gene expression analysis of human hepatocytes treated with PF-06282999 and the prototypical PXR agonist rifampin demonstrated increases in mRNA for CYP3A4 and related CYPs that are regulated by PXR.

3. Docking studies using a published human PXR crystal structure provided insights into the molecular basis for PXR activation by PF-06282999. Implementation of PXR transactivation assays in a follow-on discovery campaign should aid in the identification of back-up compounds devoid of PXR activation and CYP3A4 induction liability.     

Identification and analysis of the reactive metabolites related to the hepatotoxicity of safrole

1.?Safrole is the main component of the volatile oil in Xixin, which has a strong antifungal effect. However, safrole has been shown to be associated with the development of hepatocellular carcinoma. Methylenedioxyphenyl and allyl-benzene substructures of safrole may cause a mechanism-based inhibition (MBI) of CYP450 enzymes (CYPs) and produce reactive metabolites (RMs), resulting in inhibition of enzyme activity and toxic effects.

2.?Based on the experiments of CYPs cocktail screening, glutathione (GSH) capture and the IC₅₀ data, we found that safrole had an inhibitory effect on CYP1A2. The test of enzyme activity recovery when adding GSH may help to verify the MBI of safrole.

3.?Two metabolites, 1,2-dihydroxy-4-allylbenzene (M1) and 1′-hydroxy safrole (M2) could be captured by GSH. The ultra performance liquid chromatography - tandem mass spectrometer (UPLC-MS/MS) method was used to identify the RMs through a detailed characterization of the safrole cleavage processes and the GSH-M1 adduct. The RMs identified are quinone and its tautomer. Thus, preliminary conclusion can be obtained that safrole is a mechanism-based inhibitor of CYP1A2.

4.?The cleavage process of the GSH-M1/M2 adduct was analyzed in further detail. We believe the safrole hepatotoxicity mechanism is related to the RMs mediated by CYP1A2. This work provides important information on predicting in vivo drug induced liver injury.     

C3b inactivator deficiency: Association with an alpha-migrating factor H

Complete absence of Factor I (C3b inactivator) was found in the serum of a 3-year-old boy with recurrent polytope bacterial infections. Analysis of the complement (C) components of the patient's serum showed that while serum levels of the earlier C components (C1q, C1r, C1s, C4, and C2) were within the normal range, levels of C3, C5, Factor B, and properdin were decreased significantly. Factor H (₁H) was also decreased and, by crossed immunoelectrophoresis, migrated to the alpha region (H). C-dependent biological functions such as chemotactic activity, opsonic factors, immune adherence inhibition, and intracellular killing of bacteria were defective. The patient has been treated with fresh frozen plasma (10 ml/kg body weight) every 4 weeks for 2 years. Most of the C-derived biological functions improved, and Factor H regained beta electrophoretic mobility (H) following plasma infusion. During the period of treatment, the child has been free of bacterial infections. Mild anaphylactic reactions to the plasma occurrred on two occasions; the other 22 infusions were well tolerated.     

C1酯酶抑制剂对大鼠急性心肌缺血再灌注损伤的保护作用

目的探讨C1酯酶抑制剂对大鼠心肌缺血再灌注损伤的保护作用及其可能机制。方法将48只Wistar大鼠随机分为:假手术组、模型组、治疗组、预适应组,每组12只。建立急性心肌缺血再灌注损伤模型,模型组、治疗组、预适应组大鼠结扎左前冠状动脉30 min后再灌注2 h,假手术组不结扎。检测血清肌酸激酶(CK)、肌酸激酶同工酶(CK-MB)活性;检测各组大鼠心肌梗死范围;免疫组织化学法检测组织细胞间黏附分子1(ICAM 1)的表达及RT-PCR检测ICAM-1 mRNA的表达。结果与假手术组比较,模型组大鼠左心室收缩压、左心室压力最大上升速率及最大下降速率(±dp/dt_(max))明显降低,左心室舒张末压、CK、CK-MB、ICAM-1、ICAM-1 mRNA明显升高(P<0.01);与模型组比较,治疗组和预适应组大鼠左心室收缩压、±dp/dt_(max)明显升高(P<0.01),左心室舒张末压、CK、CK-MB、ICAM-1、ICAM-1 mRNA明显降低(P<0.05,P<0.01),心肌梗死面积明显缩小(P<0.01)。结论 C1酯酶抑制剂可能通过阻断ICAM-1的表达,从而限制中性粒细胞聚集起到心肌保护作用。     

Pharmacokinetics of trimethylamine in rats,including the effects of a synthetic diet

1. The pharmacokinetic profile of trimethylamine (TMA) was examined in the male Wistar rat and the effects of a synthetic diet on TMA pharmacokinetics were also evaluated. 2. The concentrations of TMA and its N -oxide in blood were analysed by a sensitive headspace gas chromatographic assay. 3. The pharmacokinetics of TMA were essentially linear for intravenous (i.v.) bolus doses of 10-40?mg kg -1. Over the range of administered i.v. doses, the concentrations of TMA in blood declined approximately monoexponentially with half-lives of 2.03-2.48?h. The V d of TMA ranged from 3.2 to 4.39?l kg -1 and clearance ranged from 18.78 to 23.92ml?min -1 kg -1. The peak concentration of TMA in blood occurred at 1?h after oral administration of a 20?mg kg -1 dose and the bioavailability for the oral dose averaged 81%. 4. Peak concentrations of trimethylamine N -oxide (TMAO) in blood were attained at 0.75 and 1?h after i.v and oral administration of TMA (20mg kg -1), respectively. 5. Feeding the male Wistar rat with a synthetic diet resulted in a twofold decrease in the clearance of TMA. Furthermore, the concentration of TMAO in blood after i.v. administration of TMA peaked at 1.25?h in rat placed on the synthetic diet as opposed to 0.75?h in rat placed on normal laboratory rat chow. The altered pharmacokinetic profile of TMA and its N -oxide suggest a diminished drug-elimination capacity in rat placed on the synthetic diet. 6. Dietary modulation of flavin-containing monooxygenase (FMO) activity may explain the effects of diet on the pharmacokinetics of TMA and its N -oxide.     

Metoclopramide is metabolized by CYP2D6 and is a reversible inhibitor,but not inactivator,of CYP2D6

Abstract

1. Metoclopramide is a widely used clinical drug in a variety of medical settings with rare acute dystonic events reported. The aim of this study was to assess a previous report of inactivation of CYP2D6 by metoclopramide, to determine the contribution of various CYPs to metoclopramide metabolism, and to identify the mono-oxygenated products of metoclopramide metabolism.

2. Metoclopramide interacted with CYP2D6 with Type I binding and a K_s value of 9.56?±?1.09?µM. CYP2D6 was the major metabolizer of metoclopramide and the two major products were N-deethylation of the diethyl amine and N-hydroxylation on the phenyl ring amine. CYPs 1A2, 2C9, 2C19, and 3A4 also metabolized metoclopramide.

3. While reversible inhibition of CYP2D6 was noted, CYP2D6 inactivation by metoclopramide was not observed under conditions of varying concentration or varying time using Supersomes^TM or pooled human liver microsomes.

4. The major metabolites of metoclopramide were N-hydroxylation and N-deethylation formed most efficiently by CYP2D6 but also formed by all CYPs examined. Also, while metoclopramide is metabolized primarily by CYP2D6, it is not a mechanism-based inactivator of CYP2D6 in vitro.     

Modulation of the alternative complement pathway in patients with Gram-negative bacteraemia

Summary Concentrations of C3b inactivator (C3bINA) and β1H globulin, two proteins which regluate turnover of the alternative complement pathway, have been measured in the sera of 38 patients with Gram-negative bacteraemia at the onset of fever, prior to the onset of shock. The mean serum concentrations of these two proteins was reduced compared with normal controls (t = 1.8250, P < 0.05 for C3bINA; t =3.9555, P< 0.005 for β1H These reductions were most pronounced in those patients who subsequently developed shock, with or without a fatal outcome. Serum levels of C3bINA and β1H correlated well with levels of C3 factor B, but only poorly with C4. C3bINA, but not β1H, also correlated with properdin. As a result of these findings it has been suggested that low serum concentrations of C3bINA and β1H in Gram-negative bacteraemia, may predispose to excessive alternative pathway turnover.