Role of gut in xenobiotic metabolism

The gastrointestinal tract forms the first line of defense in the body against the main load of xenobiotics. The gastrointestinal mucosa has several mechanisms through which the xenobiotics are modified. The monooxygenase activities in most species are relatively low in the mucosa as compared to the liver, but conjugation, for example, via glucuronide formation proceeds efficiently. UDP-glucuronosyltransferase activities can exceed those in the liver. Glutathione S-transferase activity is also high. The biotransformation activities are readily inducible in the mucosa and this is, at least partly, responsible for the oral-aboral gradient seen in enzyme activities. In rainbow trout glutathione S-transferase is, however, significantly higher at the aboral third than in two oral segments, although in rats the intestinal glutathione S-transferase shows a clear oral-aboral gradient. The gradient is independent of the presence of microflora at least in the case of carboxylesterase and glutathione S-transferase. A similar gradient can also be found from the gut lumen, in both germ-free and specific pathogen-free rats. The cells in the middle of the villi appear to be most responsive under the influence of inducers. The readily occurring induction in the mucosa provides a suitable model for studies on biological effects to defined compounds and mixtures.Dedicated to Professor Dr. med. Herbert Remmer on the occasion of this 65th birthday     

Role of human aldehyde dehydrogenases in endobiotic and xenobiotic metabolism

The human genome contains at least 17 genes that are members of the aldehyde dehydrogenase (ALDH) superfamily. These genes encode NAD(P)(+)-dependent enzymes that oxidize a wide range of aldehydes to their corresponding carboxylic acids. Aldehydes are highly reactive molecules that are intermediates or products involved in a broad spectrum of physiologic, biologic, and pharmacologic processes. Aldehydes are generated during retinoic acid biosynthesis and the metabolism of amino acids, lipids, carbohydrates, and drugs. Mutations in several ALDH genes are the molecular basis of inborn errors of metabolism and contribute to environmentally induced diseases.     

Role of histamine H(1) receptor in pain perception: a study of the receptor gene knockout mice

To study the participation of histamine H(1) receptors in pain perception, histamine H(1) receptor knockout mice were examined for pain threshold by means of three different kinds of nociceptive tasks. These included assays for thermal nociception (hot-plate, tail-flick, paw-withdrawal), mechanical nociception (tail-pressure), and chemical nociception (abdominal constriction, formalin test, capsaicin test) which evoked pain by the activation in nociceptive Adelta and C fibers. The mutant mice lacking histamine H(1) receptors showed significantly fewer nociceptive responses to the hot-plate, tail-flick, tail-pressure, paw-withdrawal, formalin, capsaicin, and abdominal constriction tests. Sensitivity to noxious stimuli in histamine H(1) receptor knockout mice significantly decreased when compared to wild-type mice. This data indicates that histamine plays an important role in both somatic and visceral pain perceptions through histamine H(1) receptors. The difference in the effect of histamine H(1) receptors antagonist, the active (D-) and inactive (L-) isomers of chlorpheniramine on ICR mice further substantiates the evidence of the role of histamine H(1) receptors on pain threshold.     

Role of the bradykinin B2 receptor in the maturation of blood pressure phenotype: lesson from transgenic and knockout mice

The binding of bradykinin (BK) to its B2 receptor results in a wide spectrum of biological effects including vasodilation, smooth muscle contraction and relaxation, pain, and inflammation. In order to gain a better insight into the physiological function of this potent vasoactive peptide, murine models have been created by the use of gene insertion or deletion. The results of studies using these strategies are revisited in the present article. In transgenic mice harboring the human BK B2 receptor cDNA (cHBKR), expression of the transgene was identified in the aorta, brain, heart, lung, liver, kidney, uterus and prostate gland by RT-PCR Southern blot analysis. These mice displayed an exaggerated hypotensive response to intra-aortic injection of BK, whereas the blood pressure of knockout mice, homozygous for targeted disruption of the endogenous gene, was insensitive to BK. Two transgenic mouse lines expressing the human BK B2 receptor showed a significant reduction of systolic tail-cuff blood pressure (84 +/- 1 mm Hg, n = 28; 80 +/- 1 mm Hg, n = 24; P < 0.001) compared with the control littermates (97 +/- 1 mm Hg, n = 52). Systolic blood pressure was elevated in BK B2 receptor knockout mice (124 +/- 1 mm Hg, n = 38). In heterozygous mice, systolic blood pressure was similar to that of controls until 5 month-old, then it raised to the elevated levels of knockout mice at 7 months of age. Together these data indicate that kinins acting through the B2 receptor play a role in the development of the blood pressure phenotype.     

Inter-ethnic differences in xenobiotic metabolism

The aim of safety assurance procedures is to determine a level of intake (the acceptable daily intake (ADI) or tolerable daily intake (TDI)) that is without adverse health effects in the human population. The majority of studies on inter-ethnic differences in xenobiotic metabolism have concentrated in the incidence of expression of the poor metaboliser phenotype for a number of drug metabolising enzymes. Such ethnic differences can result in different incidences of individuals at higher risk, but this would not affect the safety assurance/risk assessment outcome unless poor metaboliser status was not recognised in the database used for the initial assessment and calculation of ADI or TDI. Of far greater importance are ethnic differences which result in population differences in the mean values, and/or the extent of variability within the population, for key kinetic parameters such as the internal dose or area under the plasma concentration time curve (AUC). There are few studies on inter-ethnic differences in sensitivity and most of these relate to in vivo differences for therapeutic/pharmacological agents, so that the reported data reflect both kinetic and dynamic variability. Inter-ethnic differences are limited in extent and well within the uncertainty factor of 10 used for human variability.     

低氧信号机制的发现与肾性贫血药物治疗进展

2019年诺贝尔生理学或医学奖表彰了“细胞氧感知和适应”机制的创新发现,其相关机制与慢性肾性贫血的病理生理过程息息相关。近年来,基于氧感知通路的肾性贫血药物研发硕果累累。目前研究的重点是低氧诱导因子脯氨酰羟化酶抑制剂,主要有6种药物,均为口服制剂,可稳定升高促红细胞生成素及血红蛋白,同时可改善铁代谢,部分已经上市,部分还处于临床研究阶段。罗沙司他作为由2019年诺贝尔生理学或医学奖研究成果衍生的唯一上市的新药,是转化医学的典范,且在我国首发上市,我国在历史上首次成为全球首先批准首创作用机制药物的国家。就这一研究发现以及依据这一新机制研发的抗肾性贫血新药的研究现状进行总结。

     

Roles of histamine receptors in pain perception: a study using receptors gene knockout mice

To study the participation of histamine H1- and H2-receptors in pain perception, H1 and H2 receptor knockout (KO) mice were examined for pain threshold by means of three kinds of nociceptive tasks. These included assays for thermal, mechanical, and chemical nociception. H1KO mice showed significantly fewer nociceptive responses to the hot-plate, tail-flick, tail-pressure, paw-withdrawal, formalin, capsaicin, and abdominal constriction tests. Sensitivity to noxious stimuli in H1KO mice was significantly decreased when compared to wild-type mice. The antinociceptive phenotypes of H2KO were relatively less prominent when compared to H1KO mice. We also examined the antinociceptive effects of intrathecally-, intracerebroventricularly-, and subcutaneously-administered morphine in H1KO and H2KO mice. In these nociceptive assays, the antinociceptive effects produced by morphine were more enhanced in both H1KO and H2KO mice. The effects of histamine H1- and H2-receptor antagonists on morphine-induced antinociception were studied in ICR mice. The intrathecal, intracerebroventricular and subcutaneous co-administrations of d-chlorpheniramine enhanced the effects of morphine in all nociceptive assays examined. In addition, intrathecal co-administrations of cimetidine enhanced the antinociception of morphine in the hot plate tests. These results suggest that existing H1 and H2 receptors play an inhibitory role in morphine-induced antinociception in the spinal and supra-spinal levels.     

In silico techniques for the study and prediction of xenobiotic metabolism: A review

Knowledge about metabolism is very important to understand the health risks posed by chemicals. The biochemical process of metabolism causes activation, inactivation, toxification, detoxification as well as changes in the physicochemical properties of a chemical. The long time consumption and high costs associated with animal tests and the challenges faced by traditional quantitative structure–activity relationship (QSAR) models in dealing with situations wherein parent chemical structures are less relevant to the ultimate effects have led to the development of in silico techniques for the prediction of xenobiotic metabolism. The strengths and limitations of some of the most commonly used in silico expert systems, and their application in studying metabolism of xenobiotic chemicals, have been reviewed. The in silico metabolism simulators possessed several distinguishing features imparted in part by the nature of knowledge rules (algorithms) encoded within them and in part by the integration of QSAR libraries and computational engines.     

In silico techniques for the study and prediction of xenobiotic metabolism: a review

Knowledge about metabolism is very important to understand the health risks posed by chemicals. The biochemical process of metabolism causes activation, inactivation, toxification, detoxification as well as changes in the physicochemical properties of a chemical. The long time consumption and high costs associated with animal tests and the challenges faced by traditional quantitative structure-activity relationship (QSAR) models in dealing with situations wherein parent chemical structures are less relevant to the ultimate effects have led to the development of in silico techniques for the prediction of xenobiotic metabolism. The strengths and limitations of some of the most commonly used in silico expert systems, and their application in studying metabolism of xenobiotic chemicals, have been reviewed. The in silico metabolism simulators possessed several distinguishing features imparted in part by the nature of knowledge rules (algorithms) encoded within them and in part by the integration of QSAR libraries and computational engines.     

Anomalies in transgenic mice carrying the human interleukin-2 gene

To elucidate the in vivo function of interleukin-2 (IL-2) we produced transgenic mice with either the human genomic IL-2 gene (hIL-2) or the murine metallothionein-I promoter-human IL-2 fusion gene (MThIL-2). Nine of 12 transgenic mice independently obtained showed motor ataxia (2 mice with hIL-2, and 7 mice with MThIL-2) due to the infiltration of lymphocytes into the cerebellar tissues (Katsuki et al. 1989). In addition to the ataxia shown by both groups of transgenic mice, other features were found with respect to each introduced DNA. The transgenic mice with hIL-2 suffered from alopecia. Lymphocytic infiltrates were found in the skin underlying the hairless regions as well as in the brains. On the other hand, the male transgenic mice with MThIL-2 had atrophic testes. In these male mice, sperm or cells of the later stages of spermatogenesis were depleted in the testes without any apparent signs of an immunological response. These findings suggest that the introduced human IL-2 genes may have the pleiotropic functions of inducing tissue-specific immunological responses in the brain and skin, and of having harmful effects on spermatogenesis.     

Mammalian epoxide hydrolases in xenobiotic metabolism and signalling

Epoxide hydrolases catalyse the hydrolysis of electrophilic—and therefore potentially genotoxic—epoxides to the corresponding less reactive vicinal diols, which explains the classification of epoxide hydrolases as typical detoxifying enzymes. The best example is mammalian microsomal epoxide hydrolase (mEH)—an enzyme prone to detoxification—due to a high expression level in the liver, a broad substrate selectivity, as well as inducibility by foreign compounds. The mEH is capable of inactivating a large number of structurally different, highly reactive epoxides and hence is an important part of the enzymatic defence of our organism against adverse effects of foreign compounds. Furthermore, evidence is accumulating that mammalian epoxide hydrolases play physiological roles other than detoxification, particularly through involvement in signalling processes. This certainly holds true for soluble epoxide hydrolase (sEH) whose main function seems to be the turnover of lipid derived epoxides, which are signalling lipids with diverse functions in regulatory processes, such as control of blood pressure, inflammatory processes, cell proliferation and nociception. In recent years, the sEH has attracted attention as a promising target for pharmacological inhibition to treat hypertension and possibly other diseases. Recently, new hitherto uncharacterised epoxide hydrolases could be identified in mammals by genome analysis. The expression pattern and substrate selectivity of these new epoxide hydrolases suggests their participation in signalling processes rather than a role in detoxification. Taken together, epoxide hydrolases (1) play a central role in the detoxification of genotoxic epoxides and (2) have an important function in the regulation of physiological processes by the control of signalling molecules with an epoxide structure.

Effects of neurotensin gene knockout in mice on the behavioral effects of cocaine

Rationale  

The neuropeptide neurotensin (NT), which has been implicated in the modulation of dopamine signaling, is expressed in a subset of dopamine neurons and antagonism of the NT receptor has been reported to reduce psychostimulant-induced behavior. Gene knockout (KO) of the neurotensin/neuromedin N precursor provides an approach to delineating possible roles of endogenous NT in psychostimulant-induced responses.     

Effects of kinins on isolated stomachs of control and transgenic knockout B2 receptor mice

The aim of this study was to investigate the pharmacological profile of the kinin B₁ and B₂ receptors in isolated stomachs from wild-type control and B₂ receptor knockout mice. Isometric contractions evoked by bradykinin (BK) (9 nM) and desArg⁹BK (28 nM) were shown to be different. The contraction induced by desArg⁹BK had a longer duration than that evoked by BK and increased during incubation in vitro in stomachs of wild-type controls, while in the transgenic B₂ receptor knockout mice, the contractions evoked by desArg⁹BK and BK were similar and followed the B₁ receptor agonist pattern. BK but not the carboxypeptidase-resistant analog, [Phe⁸ψ(CH₂-NH)Arg⁹]BK, was found to be active in the stomach of B₂ receptor knockout mice. BK-induced contractions were prevented by mergetpa (a carboxypeptidase M inhibitor) (10 μM) and by a the B₁ receptor antagonist, AcLys[DβNal⁷,Ile⁸]desArg⁹BK (R 715) (0.88 μM), while not being influenced by the B₂ receptor antagonist HOE 140 (0.38 μM). BK and [Phe⁸ψ(CH₂-NH)Arg⁹]BK were potent contractants of the wild-type mice stomach and their effects were not influenced by mergetpa or by the B₁ receptor antagonist: they were reduced by HOE 140. After incubation in vitro for 3–4 hours, the tissues were treated with HOE 140 (4 μM) and FR-173657 (17 μM) to eliminate B₂ receptor function. In these tissues, BK evoked a B₁-like contraction which was inhibited by mergetpa (10 μM) and antagonized by R 715 (8 μM). The results indicate that BK acts primarily on B₂ receptors. However, after intramural conversion to desArg⁹BK, activation of B₁ receptors of the mice stomach occurs. In the tissues of B₂ receptor knockout mice, BK behaves as a pure B₁ receptor agonist while in stomachs of control animals, the B₂ receptor contribution is overwhelming. After complete blockade of the B₂ receptor, BK is able to evoke B₁-mediated responses similar to those observed in tissues of B₂ receptor knockout mice. It is concluded that the disruption of the B₂ receptor gene eliminates the B₂ receptor without influencing the B₁ receptor system. Received: 18 September 1997 / Accepted: 21 November 1997     

Computer systems for the prediction of xenobiotic metabolism

The aim of pharmaceutical research and development is to ensure a continuing pipeline of new chemical entities (NCEs) displaying high therapeutic efficacy with few or no side effects. Failure of promising lead candidates late in the drug discovery processes is regarded as commercially unacceptable in today's increasingly competitive business environment. An inappropriate ADME/Toxicity profile in humans is the major cause of failure of lead candidates in late clinical stages of drug development. Combinatorial chemistry techniques coupled with high throughput screening protocols means that pharmaceutical companies are now dealing with an unprecedented number of NCEs on an annual basis. As a consequence, screening for undesirable ADME/Toxicity properties in the early stages of drug development, preferably pre-synthesis, is now considered the essential paradigm. In silico assessment of NCEs is rapidly emerging as the next wave of technology for early ADME/Toxicity prediction. In this review, we discuss the major commercially available products for the assessing the potential metabolic activity of xenobiotic substances in mammalian systems.     

Studies on induction of lamotrigine metabolism in transgenic UGT1 mice

1. A transgenic ‘knock-in’ mouse model expressing a human UGT1 locus (Tg-UGT1) was recently developed and validated. Although these animals express mouse UGT1A proteins, UGT1A4 is a pseudo-gene in mice. Therefore, Tg-UGT1 mice serve as a ‘humanized’ UGT1A4 animal model.

2. Lamotrigine (LTG) is primarily metabolized to its N-glucuronide (LTGG) by hUGT1A4. This investigation aimed at examining the impact of pregnane X receptor (PXR), constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor (PPAR) activators on LTG glucuronidation in vivo and in vitro. Tg-UGT1 mice were administered the inducers phenobarbital (CAR), pregnenolone-16α-carbonitrile (PXR), WY-14643 (PPAR-α), ciglitazone (PPAR-γ), or L-165041 (PPAR-β), once daily for 3 or 4 days. Thereafter, LTG was administered orally and blood samples were collected over 24?h. LTG was measured in blood and formation of LTGG was measured in pooled microsomes made from the livers of treated animals.

3. A three-fold increase in in vivo LTG clearance was seen after phenobarbital administration. In microsomes prepared from phenobarbital-treated Tg-UGT1 animals, 13-fold higher CL_int (V_max/K_m) value was observed as compared with the untreated transgenic mice. A trend toward induction of catalytic activity in vitro and in vivo was also observed following pregnenolone-16α-carbonitrile and WY-14643 treatment. This study demonstrates the successful application of Tg-UGT1 mice as a novel tool to study the impact of induction and regulation on metabolism of UGT1A4 substrates.