The use of enzyme-linked immunosorbent assays (ELISA) for the determination of pollutants in environmental and industrial wastes.

Twelve enzyme-linked immunosorbent assays (ELISA), for the determination of surfactants [linear alkylbenzene sulfonates (LAS), alkyl ethoxylates (AE), and alkylphenol ethoxylates (APE)], endocrine disruptors [alkylphenol (AP), AP + APE, and bisphenol A (BPA)], estrogens [17beta-estradiol (E2), estrone (El), estrogen (ES: El + E2 + estriol (E3)), 1 7alfa-ethynylestradiol (EE2)], dioxins and polychlorinated biphenyls (PCBs), were validated on environmental water and industrial wastes. The lowest quantification limits of these ELISAs were 0.05 microg/L (BPA, E2, El, ES and EE2), 2 microg/L (AE), 3 microg/L (dioxins and PCBs), 5 microg/L (AP, AP + APE) and 20 microg/L (LAS and APE). To apply these ELISAs to environmental or industrial waste samples, simple and appropriate pre-treatment methods were also developed for each ELISA. With optimized pre-treatments, the values of ELISAs were well co-related, in all cases, to those of instrumental analytical methods such as liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and high-resolution gas chromatography mass spectrometry (HR-GC-MS), etc.

The potential of NO3--N utilization by a woody shrub species Lindera triloba: a cultivation test to estimate the saturation point of soil NO3--N for plants.

Responses of seedlings of a shrub species, Lindera triloba, grown in perlite culture medium, to nitrate (NO3--N) supply were investigated to estimate the saturating point of available NO3--N for plant utilization. NO3--N concentration and nitrate reductase activity (NRA) in leaves and roots were used as indicators of NO3--N uptake and assimilation by L. triloba. Root NRA increased with NO3--N supply when concentrations were low and reached a plateau at high NO3--N concentrations. On the other hand, root NO3--N concentration increased linearly with NO3--N supply; therefore, it is suggested that NO3--N uptake did not limit NO3--N assimilation by L. triloba. In contrast, leaf NRA and leaf NO3--N concentration were low and were not influenced by NO3--N supply. This may be caused by the lack of transport of NO3--N from roots to leaves. The NO3--N retained in perlite was compared with NO3--N pool sizes in soils from a forest where L. triloba occurs naturally to estimate the level of NO3--N availability to plants in the forest soil. The maximum NO3--N pool size in the forest soil was comparable to concentrations at which root NRA reached a plateau in perlite cultures. These results indicate that soil NO3--N availability is below the saturation point for NO3--N uptake by L. triloba, and it is the limiting factor of NO3--N utilization by L. triloba under field conditions in which this species naturally occurs.

Carrier-mediated transport of riboflavin in the rat colon.

Carriers involved in riboflavin transport have generally been presumed to be localized in the upper small intestine. However, using a closed loop technique, we found that in the rat colon the absorption of riboflavin could be significantly reduced by raising the concentration from 0.1 to 200 microM and by adding lumiflavin, an analogue of riboflavin. These results suggest that saturable transport by the carrier that is specific for riboflavin and analogues may also be involved in riboflavin absorption in the colon. At the lower concentration of 0.1 microM, carrier-mediated transport was suggested to prevail, compared with passive transport, both in the colon and the small intestine. Furthermore, carrier-mediated transport in the colon was comparable with that in the small intestine. This study is the first to suggest carrier-mediated riboflavin transport in the colon. Although the riboflavin transport system in the colon needs to be subjected to more detailed investigation of its transport functions and role in riboflavin absorption after oral administration, it would be of interest to explore potential use of this carrier as a system for drug delivery.

Superoxide dismutase activity of iron(II)TPEN complex and its derivatives.

Superoxide is involved in the pathogenesis of various diseases, such as inflammation, ischemia-reperfusion injury and carcinogenesis. Superoxide dismutases (SODs) catalyze the disproportionation reaction of superoxide to produce oxygen and hydrogen peroxide, and can protect living cells against the toxicity of free radicals derived from oxygen. Thus, SODs and their functional mimics have potential value as pharmaceuticals. We have previously reported that Fe(II)tetrakis-N,N,N',N'-(2-pyridylmethyl)ethylenediamine (Fe(II)TPEN) has an excellent SOD activity (IC50 = 0.5 microM) among many iron complexes examined (J. Biol. Chem., 264, 9243-9249 (1989)). Fe(II)TPEN can act like native SOD in living cells, and protect Escherichia coli cells from free radical toxicity caused by paraquat. In order to develop more effective SOD functional mimics, we synthesized Fe(II)TPEN derivatives with electron-donating or electron-withdrawing groups at the 4-position of all pyridines of TPEN, and measured the SOD activities and the redox potentials of these complexes. Fe(II) tetrakis-N,N,N',N'-(4-methoxy-2-pyridylmethyl)ethylenediamine (Fe(II)(4MeO)4TPEN) had the highest SOD activity (IC50 = 0.1 microM) among these iron-based SOD mimics. In addition, a good correlation was found between the redox potential and the SOD activity of 15 Fe(II) complexes, including iron-based SOD mimics reported in the previous paper (J. Organometal. Chem., in press). Iron-based SOD mimics may be clinically applicable, because these complexes are generally tissue-permeable and show low toxicity. Therefore our findings should be significant for the development of clinically useful SOD mimics.

Mechanism of superoxide dismutase-like activity of Fe(II) and Fe(III) complexes of tetrakis-N,N,N',N'(2-pyridylmethyl)ethylenediamine.

The superoxide dismutase (SOD) activity of iron(II) tetrakis-N,N,N',N'(2-pyridylmethyl)ethylenediamine complex (Fe-TPEN) was reexamined using a pulse radiolysis method. In our previous study (J. Biol. Chem., 264, 9243-9249 (1989)), we reported that this complex has a potent SOD activity in a cyt. c (cytochrome c)-based system (IC50 = 0.8 microM) and protects E. coli cells against paraquat toxicity. The present pulse radiolysis experiment revealed that Fe(II)TPEN reacts stoichiometrically with superoxide to form Fe(III)TPEN with a second-order rate constant of 3.9 x 10(6) M-1 S-1 at pH 7.1, but superoxide did not reduce Fe(III)TPEN to Fe(II)TPEN. The reaction of Fe(III)TPEN and superoxide was biphasic. In the fast reaction, an adduct (Fe(III)TPEN-superoxide complex) was formed at the second-order rate constant of 8.5 x 10(5) M-1 S-1 at pH 7.4. In the slow one, the adduct reacted with another molecule of the adduct, regenerating Fe(III)TPEN. In the cyt. c method with catalase, this Fe(III)TPEN-superoxide complex showed cyt. c oxidation activity, which had led to overestimation of its SOD activity. Based on the titration data, the main species of complex in aqueous media at neutral pH was indicated to be Fe(III)TPEN(OH-). A spectral change after the reduction with hydrated electron indicates that the OH- ion coordinates directly to Fe(III) by displacing one of the pyridine rings. The X-ray analysis of [Fe(II)TPEN]SO4 supported this structure. From the above results we propose a novel reaction mechanism of FeTPEN and superoxide which resembles a proton catalyzed dismuting process, involving Fe(III)TPEN-superoxide complex.

Novel metabolic pathway of estrone and 17beta-estradiol catalyzed by cytochrome P-450.

We have already reported that the quinol formation from some para-alkylphenols, which is a novel metabolic pathway catalyzed by cytochrome P-450, occurs in a rat liver microsomal system (). In the present study, we investigated whether estrone and 17beta-estadiol, each of which contains a p-alkylphenol moiety, are also oxidized into the corresponding quinols by cytochrome P-450. Six recombinant human cytochrome P-450 enzymes, CYP1A1, CYP1A2, CYP2B6, CYP2C9, CYP2E1, and CYP3A4, were tested. The results show that estrone and 17beta-estadiol were converted into the corresponding quinols by CYP1A1, CYP2B6, and CYP2E1.

Remarkable axial thiolate ligand effect on the oxidation of hydrocarbons by active intermediate of iron porphyrin and cytochrome P450.

In order to examine the reactivity of active intermediate derived form iron porphyrins, competitive oxidations of alkane and alkene were carried out. It has been proposed that the first step of alkane hydroxylation is H atom abstraction and that of alkene is one-electron transfer. Therefore, it is expected that alkene-alkane competitive oxidation can be used as a probe for discrimination of differences in chemical properties among active species. Cytochrome P450 and SR complex, which is a stable thiolate-ligated iron porphyrin, mediated the oxidation of alkane much more preferentially than iron porphyrin coordinated by imidazole or chloride. These results indicate that thiolate coordination alters the reactivity of the two-electron-oxidized intermediate in a manner that is much more favorable to alkane hydroxylation than the case of chloride or imidazole coordination.

Inhibition of E. coli growth by fullerene derivatives and inhibition mechanism.

Cationic ammonium fullerene derivatives (C60-bis(N, N-dimethylpyrrolidinium iodide) and C60-bis(N-methylpiperazinium iodide)) suppressed E. coli growth, whereas an anionic derivative (C60-dimalonic acid) did not. Both cationic derivatives inhibited E. coli dioxygen consumption. Inhibition of energy metabolism is concluded to be a mechanism of the growth inhibition effect of fullerene derivatives.

Dipeptides containing L-arginine analogs: new isozyme-selective inhibitors of nitric oxide synthase.

Several L-arginine analogs are known as potent inhibitors of nitric oxide synthase (NOS). We recently synthesized dipeptides containing such amino acids, and found that they are potent and isozyme-selective NOS inhibitors. For example, S-methyl-L-isothiocitrullinyl-L-phenylalanine showed 66-fold selectivity for iNOS over nNOS, while S-methyl-L-isothiocitrullinyl-L-leucine and N(G)-nitro-L-argininyl-L-phenylalanine showed 20- and 14-fold selectivity, respectively. Interestingly, S-methyl-L-isothiocitrullinyl-D-phenylalanine showed no selectivity, and S-methyl-L-isothiocitrullinyl-L-phenylalanine showed competitive inhibition. These results suggest that each NOS isozyme has a cavity of different size near the C-terminal of the L-arginine binding site, and that the selectivity of inhibitors is due to the differences in the size of the cavity.

Deprenyl decreases an endogenous parkinsonism-inducing compound, 1-benzyl-1,2,3,4-tetrahydroisoquinoline in mice: in vivo and in vitro studies.

We examined the effect of deprenyl, a promising drug for the therapy of Parkinson's disease on the formation of a parkinsonism-inducing compound, 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1BnTIQ). The 1BnTIQ content was significantly decreased in the brain of deprenyl-treated mouse in vivo, and deprenyl also inhibited 1BnTIQ formation from phenethylamine by a mouse brain homogenate supernatant in vitro. In vivo, the content of a parkinsonism-preventing compound, 1-methyl-1,2,3, 4-tetrahydroisoquinoline (1MeTIQ) was slightly increased in mice injected with deprenyl. The marked decrease of the ratio of 1BnTIQ to 1MeTIQ might play a role in the clinical effect of deprenyl.

Novel endogenous 1,2,3,4-tetrahydroisoquinoline derivatives: uptake by dopamine transporter and activity to induce parkinsonism.

We designed as candidate metabolites and synthesized two 1-benzyl-1,2,3,4-tetrahydroisoquinoline derivatives containing a dopamine moiety: 1-(3',4'-dihydroxybenzyl)-1,2,3,4-tetrahydroisoquinoline (3',4'DHBnTIQ) and 1-benzyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (6,7DHBnTIQ). Both were detected in mouse brain as endogenous amines by gas chromatography/mass spectrometry. 3',4'DHBnTIQ induced parkinsonism in mice when chronically administered intraperitoneally, whereas 6,7DHBnTIQ did not despite the structural similarity of the two compounds. This difference may be related to cellular uptake: In rat striatal synaptosomes, these compounds were intracellularly transported by the dopamine transporter with Km values of 6.14 and 7.82 microM and Vmax values of 214.3 and 112.2 pmol/min/mg of protein, respectively. Thus, endogenous 3',4'DHBnTIQ may be actively transported into dopaminergic neurons and accumulated there, contributing at least in part to the induction of idiopathic Parkinson's disease.

Real time measurement of nitric oxide released from cultured endothelial cells.

Direct detection of nitric oxide (NO) is essential for understanding the precise mechanism of its production from endothelial cells. Previously, we developed an NO detection system based on the chemiluminescence reaction between NO and luminol-H2O2. Here, we have applied this system to cultured endothelial cells for the direct and on-time measurement of NO. The perfusate from cultured endothelial cells was continuously mixed with luminol-H2O2. N(G)-monomethyl-L-arginine (L-NMMA) (10(-4) M) decreased the chemiluminescence signal of NO, suggesting the existence of basal NO release. Bradykinin (10(-8) M-10(-6) M) increased the NO signal (10(-6) M; 5.1+/-0.4 fmol/min, corresponding to 1.7 pM in the perfusate), and this was inhibited by 10(-4) M L-NMMA (1.8+/-0.3 fmol/min). These results corresponded to the changes in cGMP levels in RFL-6 cells, which provide an NO bioassay system. We conclude that the luminol-H2O2 system is useful for the direct and continuous measurement of NO from cultured endothelial cells.

Real-time measurement of nitric oxide production in rat brain by the combination of luminol-H2O2 chemiluminescence and microdialysis.

A chemiluminescence method of detecting nitric oxide (NO) in combination with a microdialysis technique was employed for the real-time measurement of NO production in living rat brain. This method based on the luminol-H2O2 system has a detection limit of 1 nM, and is the most sensitive method currently available for measuring NO. We applied this new technique to rat cerebellum to record the increase of chemiluminescence intensity arising from NO production after the injection of N-methyl-D-aspartate or kainic acid around the microdialysis probe. This highly sensitive method should be useful for the direct clarification of the functions of NO in the central nervous system.

Inhibitory effect of a fullerene derivative, monomalonic acid C60, on nitric oxide-dependent relaxation of aortic smooth muscle.

1. Effects of a fullerene C60 derivative, monomalonic acid C60 (MMA C60), on endothelium-containing or denuded aorta of rabbit, trachea and ileum of guinea pig, and stomach (fundus), vas deferens and uterus of rat were studied pharmacologically. 2. MMA C60 (10(-5) M) significantly reduced the maximum response of the relaxation induced by acetylcholine in endothelium-containing thoracic aorta of rabbit, and the acetylcholine-induced relaxation was recovered in the presence of superoxide dismutase (SOD, 250 units/ml). 3. Nitric oxide-generating agent, S-nitroso-N-acetylpenicillamine, caused the relaxation of aorta without endothelium in a concentration-dependent manner, and the concentration-response curve was shifted to the right in the presence of MMA C60. This inhibitory effect of the derivative was also masked in the presence of superoxide dismutase (SOD). 4. Sodium nitroprusside-induced relaxation was not affected by either MMA C60 or SOD. In the other tissues, this C60 derivative had no effect on the responses induced by any agonist. 5. These observations indicate that MMA C60 inhibits the endothelium-dependent relaxation induced by acetylcholine but does not affect the agonist-induced contractile response of smooth muscle.

Cross-linking of collagen by singlet oxygen generated with UV-A.

Singlet oxygen (1O2), a highly reactive and toxic intermediate, may play a role in photo-induced aging. We examined singlet oxygen generation from hematoporphyrin (HP) with UV-A, by monitoring the emission at 1,268 nm corresponding to 1O2 --> 3O2. Singlet oxygen was formed HP-dose-dependently in this system. We then investigated the reaction of singlet oxygen generated by UV-A irradiation with collagen, which is related to skin elasticity and softness. Collagen from skin was rapidly and dose-dependently cross-linked by singlet oxygen. The reaction was inhibited by NaN3, a selective quencher of singlet oxygen. In contrast, SOD (superoxide dismutase) and mannitol had no effect. These results suggested that cross-linking of collagen was caused by UV-A-generated singlet oxygen, not by any other reactive oxygen species. Compared with another multisubunit protein, alcohol dehydrogenase, collagen was cross-linked much more efficiently. Further, the finding that semicarbazide inhibited cross-linking of collagen showed that cross-links were formed between photooxidized histidyl residues and amino groups. Singlet oxygen generated by UV-A irradiation may contribute to cross-linking of collagen in the process of skin photoaging.

Participation of reactive oxygen species in phototoxicity induced by quinolone antibacterial agents.

To elucidate the mechanism of phototoxicity induced as a side effect by some of the new quinolone antibiotics, we studied sparfloxacin (SPFX), lomefloxacin, enoxacin, ofloxacin, and ciprofloxacin. We first examined the photosensitized formation of reactive oxygen species such as singlet oxygen (1O2) and superoxide anion (O2-) mediated by the new quinolones. Although a large number of studies have been reported, there is no direct evidence that these drugs generate reactive oxygen species. We employed a near-infrared emission spectrometer to detect 1O2-specific emission (1268 nm), and the nitroblue tetrazolium reduction method to detect O2-. All the quinolones investigated in this study were found to produce 1O2. Four drugs, but not SPFX, produced O2-. We also examined photodynamic DNA strand-breaking activity as a possible mechanism to explain the participation of reactive oxygen species in the phototoxicity of the drugs. All the drugs exhibited photodynamic DNA strand-breaking activity. The inhibitory effect of scavengers of reactive oxygen species indicated that the main active species was 1O2. The DNA strand-breaking activity was correlated not with the 1O2-forming ability, but with the affinity of the drugs for DNA. This result may be due to the short lifetime of 1O2. These data suggested that the phototoxicity of the new quinolones was related to DNA damage caused by reactive oxygen species, especially 1O2.

Inhibitory effects of a fullerene derivative, dimalonic acid C60, on nitric oxide-induced relaxation of rabbit aorta.

Dimalonic acid C60 (10(-5) M), a new fullerene derivative, produced an augmentation of phenylephrine-induced tone and reduced both the acetylcholine-induced maximum relaxation and the amplitude of substance P (10(-8) M)-induced relaxation in endothelium-containing thoracic aorta of rabbit; the acetylcholine- and substance P-induced relaxation was restored in the presence of superoxide dismutase (250 U/ml). Dimalonic acid C60 (10(-5) M) did not influence the phenylephrine-induced contractile response in the absence of endothelium, but the acetylcholine-induced relaxation was eliminated by removal of the endothelium. Superoxide anion generation, using hypoxanthine (1 mM)/xanthine oxidase (16 mU/ml), reduced the acetylcholine-induced relaxation and produced an augmentation of phenylephrine-induced tone in endothelium-containing strips; these effects were negated by the addition of superoxide dismutase (250 U/ml). A nitric oxide-generating agent, S-nitroso-N-acetylpenicillamine, caused relaxation of aorta without endothelium in a concentration-dependent manner, and the concentration-response curve was shifted to the right in the presence of dimalonic acid C60. This inhibitory effect of dimalonic acid C60 was also masked in the presence of superoxide dismutase. Sodium nitroprusside-induced relaxation was not affected by either dimalonic acid C60 or superoxide dismutase. These observations suggest that dimalonic acid C60 inhibits endothelium (nitric oxide)-dependent agonist-induced relaxation through the production of superoxide.

Receptor subtype for vasopressin-induced release of nitric oxide from rat kidney.

The vasopressin receptor subtype that causes nitric oxide (NO) release remains controversial. To elucidate this receptor-ligand interaction, we examined the effects of vasopressin receptor antagonists on vasopressin-induced release of NO from isolated perfused rat kidneys by using a sensitive chemiluminescence assay. Vasopressin increased renal perfusion pressure and NO signals in the perfusate in a dose-dependent manner. N omega-Monomethyl-L-arginine abolished this increase in NO release; however, a similar increase in renal perfusion pressure induced by prostaglandin F2 alpha was not associated with the increase in NO release. OPC-21268, a V1 receptor antagonist, significantly reduced the vasopressin-evoked renal vasoconstriction and NO release, whereas OPC-31260, a V2 receptor antagonist, had no effects. Moreover, desmopressin, a selective V2 receptor agonist, did not increase the NO signal. NO release by vasopressin was markedly attenuated in deoxycorticosterone acetate (DOCA)-salt hypertensive rat kidneys compared with control kidneys (10(-10) mol/L vasopressin: +0.8 +/- 0.3 versus +6.9 +/- 1.4 fmol/min per gram kidney, DOCA versus control; P < .001). Histochemical analysis for renal NO synthase revealed a substantial attenuation of the staining of endothelial NO synthase in DOCA-salt rats. These results directly demonstrate that vasopressin stimulates NO release via the endothelial V1 receptor in the rat kidney.

Substituent elimination from p-substituted phenols by cytochrome P450. ipso-Substitution by the oxygen atom of the active species.

When various p-substituted phenols (substituent = NO2, CN, CH2OH, COCH3, COPh, COOH, F, Cl, and Br) were incubated with rat liver microsomes, the substituent was eliminated to produce hydroquinone, and the reaction was inhibited by CO and a cytochrome P450-specific inhibitor. In the case of p-cresol (substituent = CH3), p-toluquinol was formed instead of hydroquinone. Experiments using 18O2 proved that the elimination is accompanied with ipso-substitution by the oxygen atom of the active species in cytochrome P450. These results are similar to those in a cytochrome P450. These results are similar to those in a cytochrome P450 chemical model system (Ohe, T., et al., Tetrahedron Lett. 42, 7681-7684, 1995), implying that the model is a good mimic of cytochrome P450. Substrates that lack a hydroxy group, namely p-substituted toluenes, did not undergo the reaction, thus indicating that a hydroxy group at the p-position to the eliminated substituent is necessary for this pathway. This is the same as the result obtained with the cytochrome P450 model. Finally, to elucidate how the substituent is eliminated, we attempted to detect the product derived from the eliminated group with several substrates. Results indicated that the mechanism of the substituent elimination can be divided into two types: the substituent is eliminated as an anion or as a cation.

Chronic administration of 1-benzyl-1,2,3,4-tetrahydroisoquinoline, an endogenous amine in the brain, induces parkinsonism in a primate.

1-Benzyl-1,2,3,4-tetrahydroisoquinoline hydrochloride (1Bn TIQHCl) (22 mg/kg per day) was subcutaneously injected into a monkey, Macaca fascicularis for 66 days to investigate its acute and chronic effects Parkinsonism like motor symptoms appeared, and the acute toxicity was stronger than the chronic toxicity. This result suggested that 1BnTIQ may induce parkinsonism in humans. 1BnTIQ content in various regions of the monkey brain was determine by the gas chromatography-selected ion-monitoring method, but no significant variation was found.