Katsuhiro Okuda

Novel Pathway of Metabolic Activation of Bisphenol A-Related Compounds for Estrogenic Activity

We previously demonstrated that estrogenic activity of bisphenol A (BPA) in the yeast estrogen screening assay was increased severalfold after incubation with rat liver S9 fraction in the presence of a NADPH-generating system. In this study, we investigated whether eight BPA-related compounds are similarly activated metabolically by rat liver S9 fraction. Three of the analogs exhibited an increase of estrogenic activity after incubation with rat liver S9 fraction but not with microsomal or cytosolic fraction alone. The structures of the metabolites formed were examined by liquid chromatography/mass spectrometry. In addition to oxidized metabolites such as catechols, we found novel dimer-type metabolites. Some of the putative metabolites were chemically synthesized to confirm their structures. The structural requirements for formation of the metabolites, some of which showed more potent estrogenic activity than the parent substrates, were examined. We have uncovered a new pathway of metabolic activation of certain phenolic compounds, such as BPA analogs, to estrogenic dimer-type compounds.

Neuroprotective effect of 1-methyl-1,2,3,4-tetrahydroisoquinoline on cultured rat mesencephalic neurons in the presence or absence of various neurotoxins

1-Methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ) is an endogenous brain amine and its content in parkinsonian brain is decreased compared with that in control brain. There is some evidence that 1MeTIQ protects dopaminergic neurons against dysfunction such as that seen in Parkinsons disease. In this study, we examined the neuroprotective effect of 1MeTIQ against four dopaminergic neurotoxins, 1-methyl-4-phenylpyridinuim ion, 6-hydroxydopamine, rotenone, and l-benzyl-1,2,3,4-tetrahydroisoquinoline, in cultured rat mesencephalic neurons. 1MeTIQ exerted neuroprotective action against all these toxins. Furthermore, (R)-1MeTIQ was neuroprotective, while (S)-1MeTIQ had little effect, indicating that the effect is stereoselective. The protective action of 1MeTIQ was most effective in mesencephalic neurons, especially in tyrosine hydroxylase-positive neurons. 1MeTIQ showed no affinity for dopamine receptors and did not influence the inhibition of mitochondrial respiratory complex I by rotenone, 1-methyl-4-phenylpyridinuim ion, or 1-benzyl-1,2,3,4-tetrahydroisoquinoline. These results raise the possibility that 1MeTIQ indirectly acts as an anti-oxidant such as the induction of anti-oxidative enzymes, because all these four neurotoxins can burden oxidative stress in common. This is the first report to confirm a protective effect of 1MeTIQ at the cultured neuron level, and it may have potential as a lead compound for the development of new agents to treat Parkinsons disease.

In vivo estrogenic potential of 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene, an active metabolite of bisphenol A, in uterus of ovariectomized rat

4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), an active metabolite of bisphenol A (BPA), has more potent estrogenic activity than BPA in vitro, but its activity in vivo is not established. Here, we examined in vivo estrogenic activity of MBP by means of uterotrophic assay in ovariectomized (OVX) female rats. MBP exhibited dose-dependent estrogenic activity, as evaluated in terms of effects on uterus weight, uterine luminal epithelial cell height and myometrium thickness. The highest concentration of MBP (10 mg/kg/day) completely reversed the changes caused by OVX, and its activity was equivalent to that of 5 microg/kg/day 17beta-estradiol (E2). We also investigated the effects of MBP on transcription of several estrogen-related genes. The changes of mRNA levels of estrogen receptors alpha and beta, c-fos and insulin-like growth factor 1 in MBP-treated OVX rats were qualitatively similar to those in E2-treated rats. BPA did not show any significant effect on OVX rat in these conditions. This study is the first to demonstrate that MBP, an active metabolite of BPA, has potent in vivo estrogenic activity, being about 500-fold more potent than BPA in OVX rats.

Tributyltin-induced endoplasmic reticulum stress and its Ca2 +-mediated mechanism

Organotin compounds, especially tributyltin chloride (TBT), have been widely used in antifouling paints for marine vessels, but exhibit various toxicities in mammals. The endoplasmic reticulum (ER) is a multifunctional organelle that controls post-translational modification and intracellular Ca(2+) signaling. When the capacity of the quality control system of ER is exceeded under stress including ER Ca(2+) homeostasis disruption, ER functions are impaired and unfolded proteins are accumulated in ER lumen, which is called ER stress. Here, we examined whether TBT causes ER stress in human neuroblastoma SH-SY5Y cells. We found that 700nM TBT induced ER stress markers such as CHOP, GRP78, spliced XBP1 mRNA and phosphorylated eIF2α. TBT also decreased the cell viability both concentration- and time-dependently. Dibutyltin and monobutyltin did not induce ER stress markers. We hypothesized that TBT induces ER stress via Ca(2+) depletion, and to test this idea, we examined the effect of TBT on intracellular Ca(2+) concentration using fura-2 AM, a Ca(2+) fluorescent probe. TBT increased intracellular Ca(2+) concentration in a TBT-concentration-dependent manner, and Ca(2+) increase in 700nM TBT was mainly blocked by 50μM dantrolene, a ryanodine receptor antagonist (about 70% inhibition). Dantrolene also partially but significantly inhibited TBT-induced GRP78 expression and cell death. These results suggest that TBT increases intracellular Ca(2+) concentration by releasing Ca(2+) from ER, thereby causing ER stress.

Neuroprotective or neurotoxic activity of 1-methyl-1,2,3,4-tetrahydroisoquinoline and related compounds.

1-Methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ) 1 and various 5- or 6,7-substituted analogues were synthesized and assayed for neurotoxicity towards SH-SY5Y cells. Among mono-substituted derivatives of 1, hydroxyl substitution decreased the toxicity, while methoxyl substitution increased it. Disubstituted derivatives of 1, 5a and 5b, showed the opposite tendency. Hydroxy-1MeTIQ derivatives were tested for neuroprotective activity, and 3b and 4b exhibited greater efficacy than 1. We suggest that hydroxy-1MeTIQ derivatives, especially 4b, may have potential for the treatment of Parkinsons disease.

Endogenous neurotoxic dopamine derivative covalently binds to Parkinson's disease‐associated ubiquitin C‐terminal hydrolase L1 and alters its structure and function

Parkinsons disease (PD) is a common neurodegenerative disease, but its pathogenesis remains elusive. A mutation in ubiquitin C‐terminal hydrolase L1 (UCH‐L1) is responsible for a form of genetic PD which strongly resembles the idiopathic PD. We previously showed that 1‐(3′,4′‐dihydroxybenzyl)‐1,2,3,4‐tetrahydroisoquinoline (3′,4′DHBnTIQ) is an endogenous parkinsonism‐inducing dopamine derivative. Here, we investigated the interaction between 3′,4′DHBnTIQ and UCH‐L1 and its possible role in the pathogenesis of idiopathic PD. Our results indicate that 3′,4′DHBnTIQ binds to UCH‐L1 specifically at Cys152 in vitro. In addition, 3′,4′DHBnTIQ treatment increased the amount of UCH‐L1 in the insoluble fraction of SH‐SY5Y cells and inhibited its hydrolase activity to 60%, reducing the level of ubiquitin in the soluble fraction of SH‐SY5Y cells. Catechol‐modified UCH‐L1 as well as insoluble UCH‐L1 were detected in the midbrain of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine‐treated PD model mice. Structurally as well as functionally altered UCH‐L1 have been detected in the brains of patients with idiopathic PD. We suggest that conjugation of UCH‐L1 by neurotoxic endogenous compounds such as 3′,4′DHBnTIQ might play a key role in onset and progression of idiopathic PD.

Determination method of 1-methyl-1,2,3,4-tetrahydroisoquinoline, an endogenous parkinsonism-preventing substance, by radioimmunoassay

To develop a sensitive and simple assay method for 1-methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ), an endogenous parkinsonism-preventing substance, we designed two kinds of 1MeTIQ-bovine serum albumin (BSA) conjugates to recognize the methyl group at the 1 position of 1MeTIQ since this is the critical structural difference between 1MeTIQ and parkinsonism-inducing substances. These hapten antigens were synthesized from 1MeTIQ analogues and BSA. A specific antiserum against 1MeTIQ was obtained from a rabbit immunized with one of the hapten antigens. To utilize this antiserum for radioimmunoassay, detailed studies were carried out to establish optimum conditions. The antiserum recognized 1MeTIQ and showed little cross-reactivity with endogenous 1MeTIQ analogues and proteins. It was confirmed to be suitable for radioimmunoassay, and a standard curve was prepared in the range of 0.5 to 100 pmol of 1MeTIQ. This method was sensitive enough to measure endogenous 1MeTIQ in rat brain. This method should be applicable for evaluation of the progression or prognosis of Parkinsons disease (PD).

Development of a simple measurement method for GluR2 protein expression as an index of neuronal vulnerability

In vitro estimating strategies for potential neurotoxicity are required to screen multiple substances. In a previous study, we showed that exposure to low-concentrations of some chemicals, such as organotin, decreased the expression of GluR2 protein, which is a subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors, and led to neuronal vulnerability. This result suggested that GluR2 decreases as an index of neuronal cell sensitivity and vulnerability to various toxic insults. Accordingly, we developed a versatile method that is a large scale determination of GluR2 protein expression in the presence of environmental chemicals by means of AlphaLISA technology. Various analytical conditions were optimized, and then GluR2 protein amount was measured by the method using AlphaLISA. The GluR2 amounts were strongly correlated with that of measured by western blotting, which is currently used to determine GluR2 expression. An ideal standard curve could be written with the authentic GluR2 protein from 0 ng to 100 ng. Subsequently, twenty environmental chemicals were screened and nitenpyram was identified as a chemical which lead to decrease in GluR2 protein expression. This assay may provide a tool for detecting neurotoxic chemicals according to decreases in GluR2 protein expression.

Development for the measurement of serum thiosulfate using LC–MS/MS in forensic diagnosis of H2S poisoning

Thiosulfate measurement is crucial to diagnosis of hydrogen sulfide (H2S) poisoning in forensic toxicology. Although GC-MS method is currently regarded as a standard thiosulfate measurement, it requires complicated sample preparation prior to analysis. This study presents a simple, rapid, and highly sensitive method for the quantitative analysis of serum thiosulfate by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This method is based on selected reaction monitoring and has high sensitivity with a lower quantification limit of 0.5μM. Precision and accuracy of this method meet the basic requirements for quantitative analysis (intra- and inter-day tests have a relative standard deviation of ⩽10.4%; range of analytical recovery is 94.3-102.6%). On the measurements of serum thiosulfate by our developed method, a thiosulfate concentration as 57.5μM was detected clearly in the H2S poisoning case comparing to the non poisoning case in which only a trace amount of thiosulfate was observed.

Liquid chromatography-tandem mass spectrometry method for the determination of thiosulfate in human blood and urine as an indicator of hydrogen sulfide poisoning

Being a stable metabolite of hydrogen sulfide, thiosulfate has been utilized as an index for hydrogen sulfide poisoning (HSP). Thiosulfate analysis is mainly performed using gas chromatography/mass spectrometry (GC-MS) due to its high sensitivity and specificity. The GC-MS analysis requires two-step derivatizations of thiosulfate, and the derivative is not stable in solution as it has a disulfide moiety. To resolve this stability issue, we developed a novel analytical method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for monitoring the pentafluorobenzyl derivative of thiosulfate (the first reaction product of the GC-MS method) in this study. The established method exhibited high reproducibility despite being a more simplified and rapid procedure compare to the GC-MS method. Phenyl 4-hydroxybenzoate was used as an internal standard because 1,3,5-tribromobenzene which had been used in the GC-MS method was not suitable compound for LC-MS/MS with Electrospray ionization (ESI) negative detection. The linear regression of the peak area ratios versus concentrations was fitted over the concentration ranges of 0.5-250μM and 0.25-250μM in blood and urine, respectively. The validation results satisfied the acceptance criteria for intra- and inter-day accuracy and precision. Blood and urine samples from 12 suspected HSP cases were tested using this method. The thiosulfate concentration detected in the sample coincided well with that determined at the scene of each HSP accident.