Abuse of drugs remains a serious and world-wide problem. As exemplified by opioids, there are large inter-individual differences in the effects and toxicity of drugs. However, the mechanism underlying such inter-individual differences remains largely unknown. Since, in many cases, drug metabolism affects the effects of drugs, the different metabolic capacity of individuals may explain these inter-individual differences. In this review, we shall focus on drugs of abuse and survey briefly their metabolism and the enzymes involved. In addition, we shall discuss the putative origin of the inter-individual differences in sensitivity to drugs of abuse.
Far UV circular dichroism (CD) and fluorescence spectroscopy were used to investigate the interaction between bovine serum albumin (BSA) and metallothionein (MT). Both spectroscopic probes gave proofs on the interaction of the two proteins. At pH 4.0, 7.0 and 9.0, BSA showed a negative increase in ellipticity at the far-UV range in the presence of MT indicating an increase in a-helical content and a decrease in β-sheet structure. In the presence of MT at pH 4.0 and 9.0, a decrease in fluorescence intensity was observed. Tryptophan fluorescence quenching experiments were also performed using acrylamide and KI as quenchers. Under acidic conditions, a four-fold increase in Stern-Volmer constant (KSV) was observed for BSA + MT. At neutral and basic conditions, a decrease in KSV values were observed which indicates conformational changes in BSA upon binding MT. These changes are close to the region where the tryptophan residues are located in the protein.
To clarify the degradation pathway of acrinol by light, isolation and identification of acrinol degradation products (ANDP) were attempted. A novel acrinol degradation product, ANDP-7, one of the ANDPs, was isolated by extraction with methanol from cloths dampened with acrinol solution, and purified by column chromatography on Diaion HP-10 and Sephadex LH-20. The structural elucidation of ANDP-7 was examined by 1H-NMR, 13C-NMR and EI-mass spectra sudies. From the spectroscopic data, the structure of ANDP-7 was determined to be 9-amino-7-ethoxypyrrolo[3,4-b]quinoline-1,3-dione, that was found to be a novel degradation product of acrinol by light. Antimicrobial activities of ANDP-7 against Gram-positive and -negative bacteria were 5 to 100-fold higher than those of acrinol. ANDP-7 was also active against yeast and fungi. Nevertheless, acrinol did not show growth inhibition even at a concentration 100 μg/ml.
Most, if not all, of physiological and behavioral processes exhibit circadian rhythms. Recently, molecular clocks similar to those operating in the suprachiasmatic nucleus (SCN) neurons have been found in several peripheral tissues. Among peripheral tissues, several recent studies have revealed that white adipose tissues secrete a number of biologically active molecules such as leptin, resistin, adiponectin, etc. Importantly, the levels of these cytokine-like molecules are associated with development of lifestyle-related diseases such as diabetic mellitus, cardiovascular diseases, and obesity. In this study, we attempted to characterize circadian gene expression in adipose tissue. Here, we show that the expression of several clock genes exhibits daily oscillation in mice white adipocytes. Circadian expression of adipocytes-related genes such as peroxisome proliferator-activated receptor (PPAR) γ2 was also observed. Interestingly, expression pattern of some clock genes in adipocytes is distinct from that in stromal-vascular fractions containing preadipocytes. In addition to these in vivo studies, we demonstrated that serum- or dexamethasone-shock induced the cyclic gene expression of clock genes in cultured adipocytes. Consequently, we are led to conclude that adipocytes contain the machineries necessary for circadian oscillation similar to that found in SCN. We then examined that whether the pharmacological effects of PPARγ2 ligand depend on the time of administration. Consist with its receptor levels, the pharmacological effects of PPARγ ligand administered during a dark period were more efficient than those during a light period in mice. These results suggest that chronotherapy targeted for adipocyte functions could be effective in improving of the symptoms of hyperlipidemia and other related diseases.
Irregular meals (mainly the skipping of breakfast) and inadequate sleep has become prevalent in young people. However, there has been inadequate attention on the adverse effects of this nocturnal life. We observed the 24-hr patterns of sex hormones and other relative hormones in ten medical students, with the randomized cross-over design, on either a diurnal life or a nocturnal life. The subjects on a diurnal life ate three meals (07:00, 13:00 and 19:00) and slept from 22:30 to 06:30. Nocturnal life was designed by skipping their breakfast but consuming a lot (> 50% of their daily food intake) in the evening and at night, sleeping from 01:30 to 08:30 the next morning. After three weeks in the experimental life, the 24-hr plasma concentrations of testosterone, free testosterone, dihydrotestosterone (DHT), androstenedione, prolactin, estrone, luteinizing hormone, cortisol and sex hormone binding globulin (SHBG) were measured every three hours. Plasma free testosterone and prolactin decreased, but cortisol increased when subjects followed the nocturnal life. In the diurnal lifestyle group, circadian rhythms were observed in the androgens (testosterone, free testosterone, DHT, androstenedione), prolactin, cortisol and SHBG in the cosinor analysis; however, circadian rhythms were almost extinguished in the nocturnal lifestyle group. Since sex hormones play important roles in the normal physiological condition, the effects of these changes in hormone concentration and its circadian rhythm should be considered and studied intensively.
This paper describes the deficits in brain regional growth of rats treated with methylmercury (MeHg) among the postnatal developing phases. Rats were orally administered 10 mg/kg/day of methylmercury chloride (MMC) for 10 consecutive days from postnatal days 1 (PD-1), -14 and -35, which corresponded to the early-, late- and post-brain growth spurt, respectively. Weight-matched control rats were periodically isolated from their mother or diet and placed in an incubator for intervals of 4 to 10 hr in order to adjust the body weight to MMC-treated rats. The earlier the postnatal phase the higher the resistance to body weight loss induced by MMC. The rats were dissected on the day after final MMC treatment and the weight of organs and their mercury (Hg) concentrations were measured. Hg accumulation in the brain on the day after final treatment with 10 mg/kg/day of MMC was highest in the rats treated during the late-brain growth spurt. On the other hand, Hg accumulations in the liver and kidney increased rapidly with development of postnatal phases. Then, the brain/kidney and brain/liver ratio of Hg concentration were much higher in early postnatal rats than in later one. The weight of brain regions in MMC-treated rats was compared with those in weight-matched control rats. The significantly lower weight of the cerebrum, cerebellum and midbrain + diencephalon were confirmed in rats treated with MMC during the early-brain growth spurt. The significantly lower cerebellum weight was confirmed in rats treated with MMC during the late-brain growth spurt. The Significant differences were not observed in the brain regions in rats treated during post-brain growth spurt. In the case of human, a similar reduction of the brain weight occurred in the fetal and non-fetal infantile Minamata disease patients. The experiment using postnatal rats succeeded to reproduce the deficit in the brain growth during the early- and late brain growth spurt by MMC treatment.
The estrogenic and anti-androgenic activities of benzophenone and 19 hydroxylated derivatives were measured in reporter gene assays using transfected human estrogen and androgen receptors in Chinese hamster ovary cells. Eighteen benzophenones had estrogenic activity and seventeen also had anti-androgenic activity. In both assays, 2,4,4′-trihydroxybenzophenone and 2,2′,4,4′-tetrahydroxybenzophenone showed the strongest activity which were comparable to bisphenol A or 1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene (DDE). The structure-activity relationships of the estrogenic activity in this mammalian reporter gene assay were mostly similar to the yeast two-hybrid assay as previously reported. Benzophenones hydroxylated at the 3 or 4-position showed the estrogenic activity, while the others showed negative or weakly positive activities. Moreover, a hydroxyl group added at the 2-position of the 4-hydroxylated benzophenone enhanced activity, but reduced activity at the 3-position. In contrast, different results were obtained when a hydroxyl group was added to another benzene ring. The added hydroxyl group enhanced the activity in this reporter gene assay, but reduced it in the yeast two-hybrid assay. Results from the reporter gene assay corresponded with the in vivo uterotrophic assay. On the other hand, a hydroxyl group at the 2-position generally enhanced the anti-androgenic activity, though the effect of other hydroxyl groups was less clear. Meanwhile, these benzophenones had no or very weak androgen agonistic activities.
We examined the immune response of CD4-mutant mice that have soluble form of CD4 in the circulation without expression of CD4 on T cell surface. We infected the CD4-mutant mice of BALB/c and C57BL/6 backgrounds with Leishmania major and subsequently examined parameters of disease and immune response. In contrast to wild-type (wt) BALB/c mice, mutant mice of both strains showed decreased parasite replication and an intense leishmanial antigen-specific delayed-type hypersensitivity (DTH) response. In vitro cytokine analysis revealed that popliteal lymph node cells (LNC) from mutant mice of both strains secreted little or no detectable interleukin-4 (IL-4) and they secreted interferon-γ (IFN-γ) at levels similar to those of wt mice. The cytokine profile shows that a lack of IL-4 production underlies the lack of T helper type 2 (Th2) response. As validation of the impaired Th2 response, infection of the mice with Nippostrongylus brasiliensis, a typical Th2 inducer, showed a lack of Th2 response. Furthermore, in contrast to LNC from wt mice, Th2 cells were not induced from naive LNC of mutant mice when the cells were cultured in the presence of IL-4 and anti-IL-12 antibody. These findings indicate that the lack of IL-4 production is due to a lack of CD4 on T cells and that IFN-γ production is independent of CD4 on T cells. Thus, Th1 and Th2 responses to leishmanial infection are not due to the balance of IL-4/IFN-γ that are produced, but the production of IL-4 determines whether a Th1 or Th2 response will develop.
The effects of estrogenic compounds on nitric oxide (NO) production by macrophages were examined. 17β-Estradiol promoted NO production triggered by lipopolysaccharide (LPS) and/or interferon (IFN)-γ in the mouse macrophage cell line J774.1. Other estrogen-like substances such as estrone, 17α-ethynylestradiol and bisphenol A also enhanced NO synthesis, but this NO synthesis was not activated by Ca2+ ionophore A23187. RT-PCR analysis demonstrated induction of inducible nitric oxide synthase (iNOS) mRNA in J774.1 cells exposed to 17β-estradiol. Although the estrogen receptor (ER)-antagonist ICI-182780 partially suppressed the promoting effect of 17β-estradiol on NOS activity, there was little ERα detectable by RT-PCR from J774.1 cells. These results suggest that ERs may participate only partially in iNOS mRNA transcription in J774.1 cells and that 17β-estradiol may act directly through other unknown intracellular signal transduction(s) that are activated by LPS and IFN-γ.
An investigation of the metal concentrations of calcium, copper, iron, magnesium, and zinc in three groups of young female hair (n = 180), from women aged between 15 to 19 years, with different body mass indexes (BMI) of BMI < 18, BMI between 18 and 23, and BMI > 24 was performed using atomic absorption spectroscopy (AAS). The hair samples were washed with organic solvent (normal hexane : ethyl alcohol : acetone, v/v, 4 : 2 : 1) to remove the external contents and then digested with a microwave digester. The hair samples were analyzed using a flame atomic absorption spectrophotometer. The BMI < 18 group (n = 45) had the highest concentrations of calcium, copper, iron, magnesium and zinc while the BMI between 18 and 23 group (n = 75) had the second highest concentrations. The BMI > 24 group (n = 60) had the lowest concentrations. Further -more, when we compared the concentrations of calcium, copper, iron, magnesium, and zinc between the groups with BMI < 18 and BMI > 24, there are significant differences (p < 0.05) in the calcium, copper, magnesium, and zinc concentrations between these two groups. The difference was especially significant (p < 0.01) in iron concentration.
Both the glucocorticoid receptor (GR) agonist dexamethasone (DEX) and GR antagonist pregnenolone 16α-carbonitrile (PCN) enhanced the transcription of CYP3A1 in rats though in a different fashion. Seven-week old male Wister rats were intraperitoneally administered one to three times with corn oil (vehicle) or DEX and/or PCN (80 mg/kg each) at 24-hr intervals according to various schedules. The animals were sacrificed twenty-four hours after the last treatment, and the dissected livers and lungs were examined for mRNA contents of CYP3A1 and pregnane X receptor (PXR) by real time PCR. DEX appeared to act mainly as an inducer of PXR, that in turn transactivated the CYP3A1 gene, by activating the GR, while PCN was deduced to have a direct effect on the expression of the CYP3A1 gene via activation of PXR from the rapid increase in CYP3A1 mRNA in comparison with DEX. Sequential treatment with DEX and PCN in this order induced CYP3A1 mRNA expression more effectively than treatment in the opposite order. When DEX and PCN were administered simultaneously, the induction of PXR by DEX was reversed by PCN, resulting from their competitive effects on the GR. In the lung, an increase in the CYP3A1 mRNA level was observed in the presence of DEX but not PCN, independently of the GR-activation.
The measurements of organochlorine pesticides in mussels and oysters collected along the east coast of Thailand indicate that DDTs, chlordanes (CHLs), hexachlorocyclohexanes (HCHs) and hexachlorobenzene (HCB) are the main organochlorine residues. The results of the study revealed that DDT residues were present in the highest concentration in green mussels and oysters, followed by CHLs, HCHs, and HCB. However, the residue levels in the present study were much lower than those found elsewhere in the tropical coastal water of Asia. Polychlorinated biphenyls (PCBs) were present also in high concentrations in urban and industrial areas. It is interesting that tetra-, penta-, hexa- and hepta-chlorinated isomers were the predominant homologues in both the mussel and oyster samples. The PCB profile in the sediment column from the study area, reflects the active use of PCBs along the east coast of Thailand during the period of 1972-1992. Considering the low levels of organochlorine pesticides (OCs) and PCBs in green mussels and oysters found in this study, the contamination from these organic pollutants in the study area is not particularly serious in the view of human health and ecosystem perspectives.
The protective effects of green tea polyphenols on nitrogen oxide (NOx)-induced sister chromatid exchange (SCE) in cultured cells were studied. (-)Epigallocatechin gallate (EGCG) and (+)catechin (CT), the major polyphenol constituents of green tea, were found to reduce the frequency of SCE induced by NOx. NO releaser (NOR4)-released NO and NaNO2 were used as NOx. Significantly fewer SCEs were induced by 30 μM NOR4 and NaNO2 after the treatment of cells with 0.1 μM EGCG or 0.5 μM CT. NOx (NO, NO2-) is a potent environmental pollutant owing to its carcinogenic properties. Therefore these experimental results indicate the protective effects of green tea against NOx-type carcinogens.
We investigated the estrogenic activity of a bisphenol A (BPA) metabolite (4-methyl-2,4-bis(p-hydroxyphenyl)-pent-1-ene; MBP) in male medaka (Oryzias latipes) using vitellogenin (Vg, Vg1 and Vg2) as a biomarker. Male d-rR medaka were exposed to various concentrations of estradiol-17β (E2), MBP and BPA for 3 days, and then the serum Vg concentration was measured using specific chemiluminescent immunoassays. The estimated relative estrogenic activities of MBP and BPA compared with E2 (100%) were 1.3-1.4% and 0.00010-0.00023%, respectively. These findings indicated that MBP has about 104-fold higher estrogenic potency than the parent BPA and about 1/50 that of E2 for Vg synthesis in medaka. This is the first study to show that MBP can act as a highly potent estrogen agonist in living organisms.
The amount of retrovirus infection depended on the temperature during the virus entry period. Higher amount of retrovirus [human immunodeficiency virus type-1 (HIV-1) and murine leukemia virus] infected to cell at 40°C than at 37°C. One-hour adsorption at 40°C marked more than 10 times large amount of infection than that at 37°C. This phenomenon is observed in both X4 and R5 type HIV-1 strains. At 40°C condition, more than four times much provirus DNA was accumulated in cell than that at 37°C condition, while same amount of virus adsorption was observed in both at 40 and 37°C adsorption. Similar enhancement was observed in murine leukemia virus infection, while little effect was monitored in rhabdovirus (VSV) infection. This result said that high temperature condition prompted the retrovirus penetration (membrane fusion mediated way) to cell. This observation would applicable to improve the gene transduction efficiency using retrovirus vector.
The blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) work effectively to restrict the free exchange of compounds between blood and brain. It is well known that P-glycoprotein (P-gp) that is expressed in brain microvessel endothelial cells contributes to the BBB as an efflux transporter. Western blot analysis has shown that P-gp is also expressed in the choroid plexus forming the blood-CSF barrier. The purpose of this study was to examine the role of P-gp in the efflux transport of its substrates across the blood-CSF barrier. The CSF concentration of etoposide and digoxin after intracerebroventricular administration, and the CSF concentration of 99mTc-sestamibi after intravenous administration were compared between wild-type and P-gp knockout mice. After intracerebroventricular administration, there was no significant difference in the remaining concentration of etoposide and digoxin in CSF between wild-type and P-gp knockout mice, respectively. After intravenous administration of 99mTc-sestamibi, its CSF-to-serum concentration ratio in P-gp knockout mice was not significantly different from that in wild-type, whereas its brain-to-serum concentration ratio was significantly increased in P-gp knockout mice compared with wild-type mice (1.62-fold, p < 0.05). In addition, immunohistochemical analysis showed a low membrane expression of P-gp in mouse choroid plexus, compared with brain microvessels. These results suggest that P-gp does not play an important role in the transport of its substrates across the blood-CSF barrier.
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