Increase in in utero exposure to a migrant, 4,4'-butylidenebis(6-t-butyl-m-cresol), from nitrile-butadiene rubber gloves on brain aromatase activity in male rats.

4,4'-Butylidenebis(6-t-butyl-m-cresol) (BBBC) can be eluted from disposable gloves made of nitrile-butadiene rubber and possibly also detected in food. It has been reported that BBBC is an androgen and estrogen antagonist in vitro. Previously, BBBC (1.0 mg/kg body weight (bw)/d) was subcutaneously administered to pregnant rats from gestation days 11 through 18, and the effects on male offspring (postnatal day 102) were examined. Altered levels and turnover of the monoamines dopamine, serotonin, and noradrenalin as well as their metabolites were detected. This report measured the level of serum testosterone following prenatal exposure to BBBC (0.1, 1.0, 10 mg/kg bw/d) in male rats, and measured aromatase activity of the hypothalamus-preoptic area with a close connection to the sexual differentiation and sexual behavior of BBBC-treated rat brains. The serum testosterone level rose depending on exposure, and aromatase activity of the basomedial nucleus of amygdale region was increased in the BBBC-treated group compared with the control. These results suggested that prenatal exposure to BBBC affects the central nervous system of male rat offspring, and BBBC may be an endocrine disrupting-chemical during the fetal period, and might influence the functional development of the brain.

The effects of in utero exposure to a migrant, 4,4'-butylidenebis(6-t-butyl-m-cresol), from nitrile-butadiene rubber gloves on monoamine neurotransmitter in rats.

4,4'-Butylidenebis(6-t-butyl-m-cresol) (BBBC) can be eluted from disposable gloves made of nitrile-butadiene rubber and possibly also detected in food. We have reported that BBBC is an androgen and estrogen antagonist. In this report, BBBC (0.1, 1.0 mg/kg body weight (bw)/day) was subcutaneously administered to pregnant rats from gestation days 11 through 18 and the effects on male offspring (postnatal day (PND) 102) were examined. Body weight at lactation and brain weight at PND 102 were decreased in the 1.0 mg/kg bw BBBC-treated group. Altered levels and turnover of the monoamines dopamine (DA), serotonin (5-HT), and noradrenalin (NA) as well as their metabolites were detected. In the prefrontal cortex DA, 3,4-dihydroxyphenylacetic acid (DOPAC), 5-HT, 5-hydroxyindole-3-acetic acid (5-HIAA) levels were significantly increased, but homovanillic acid (HVA)/DA was decreased. In the striatum NA level, DOPAC/DA and HVA/DA were significantly increased, but 3-methoxy-4-hydrophenylglycol hemipiperazinium (MHPG) level and MHPG/NA were decreased. In hippocampus MHPG level was significantly decreased. In hypothalamus 5-HIAA level and 5-HIAA/5-HT were significantly increased. These results suggested that prenatal exposure to BBBC affects the central nervous system of male rat offspring, and BBBC may be an endocrine disrupting-chemical during the fetal periods.

[In-vitro screening of psychoactive drugs to prevent abuses].

The Tokyo Metropolitan Government in Japan enacted an "Ordinance concerning the abuse prevention of the psychoactive drugs" in April 2006 that prohibited the manufacture, cultivation, sales, possession, use, etc., of these drugs. Therefore, we constructed a reproducible, simple, and small-scale determination method of the psychoactive drugs for the re-uptake and the release of monoamines (dopamine, serotonin and norepinephrine), and the activation of [(35)S]guanosine-5'-O-(3-thio)-triphosphate binding to guanine nucleotide-binding proteins (G proteins). These assays were then applied to study the effects of different kinds (phenethylamine derivatives, tryptamine derivatives, and piperazine derivatives) of non-medically used psychoactive drugs on monoamine re-uptake and release, and G-protein binding. The results suggested that some drugs strongly act on the central nerve system to the same extent as the drugs. This assay system was able to designate psychoactive drugs as prohibited substances in accordance with criteria set forth by the Tokyo Metropolitan government.

Endocrine disruptive effects of chemicals eluted from nitrile-butadiene rubber gloves using reporter gene assay systems.

Disposable gloves made of nitrile-butadiene rubber (NBR) are used for contact with foodstuffs rather than polyvinyl chloride gloves containing di(2-ethylhexyl)phthalate (DEHP), because endocrine-disruptive effects are suspected for phthalate diesters including DEHP. However, 4,4'-butylidenebis(6-t-butyl-m-cresol) (BBBC), 2,4-di-t-butylphenol, and 2,2,4-trimetyl-1,3-pentanediol diisobutyrate can be eluted from NBR gloves, and possibly also detected in food. In this study, we examined the endocrine-disrupting effects of these chemicals via androgen receptor (AR) and estrogen receptor (ER)-mediated pathways using stably transfected reporter gene cell lines expressing AR (AR-EcoScreen system) and ER (MVLN cells), respectively. We also examined the binding activities of these chemicals to AR and ER. The IC50 value of BBBC for antagonistic androgen was in the range of 10(-6)M. The strength of inhibition was about 5 times that of a known androgen antagonist, 1,1'-(2,2-dichloroethylidene)bis[4-chlorobenzene] (p,p'-DDE), and similar to that of bisphenol A. The IC50 value of BBBC for antagonistic estrogen was in the range of 10(-6)M. These results suggest that BBBC and its structural homologue, 4,4'-thiobis(6-t-butyl-m-cresol) are androgen and estrogen antagonists. It is therefore necessary to study these chemicals in vivo, and clarify their effect on the endocrine system.

In vitro screening of psychoactive drugs by [(35)S]GTPgammaS binding in rat brain membranes.

We constructed a reproducible, simple, and small-scale determination method of the psychoactive drugs that acted directly on the monoamine receptor by measuring the activation of [(35)S]guanosine-5'-O-(3-thio)-triphosphate binding to guanine nucleotide-binding proteins (G proteins). This method can simultaneously measure the effects of three monoamines, namely dopamine (DA), serotonin (5-HT), and norepinephrine (NE), in rat brain membranes using a 96-well microplate. Activation of D(1) and D(2) receptors in striatal membranes by DA as well as 5-HT and NEalpha(2) receptors in cortical membranes could be measured. Of 12 tested phenethylamines, 2,5-dimethoxy-4-chlorophenethylamine (2C-C), 2,5-dimethoxy-4-ethylphenethylamine (2C-E), and 2,5-dimethoxy-4-iodophenethylamine (2C-I) stimulated G protein binding. The other phenethylamines did not affect G protein binding. All 7 tryptamines tested stimulated G protein binding with the following rank order of potency; 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT)>5-methoxy-N,N-diallyltryptamine (5-MeO-DALT)>5-methoxy-alpha-methyltryptamine (5-MeO-AMT)>or=5-methoxy-N,N-methylisopropyltryptamine (5-MeO-MIPT)>5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT)>N,N-dipropyltryptamine (DPT)>or=alpha-methyltryptamine (AMT). This assay system was able to designate psychoactive drugs as prohibited substances in accordance with criteria set forth by the Tokyo Metropolitan government.

Effects of oseltamivir phosphate (Tamiflu) and its metabolite (GS4071) on monoamine neurotransmission in the rat brain.

As abnormal behaviors such as jumping and falling from balcony were reported in patients aged 10 to 19 years who administrated oseltamivir phosphate (Tamiflu) for treatment influenza infection, the Ministry of Health, Labor and Welfare in Japan notified that, as a rule, Tamiflu should not be prescribed to patients aged 10 to 19 years. To examine the relationship between Tamiflu and abnormal behaviors, we investigated the effects of Tamiflu and its carboxylic acid metabolite, GS4071, on the central nervous system, that is, on 3 neurotransmitters (dopamine, serotonin, and norepinephrine) in presynapses (inhibition of re-uptake, promotion of release) and postsynapses (guanosine 5'-triphosphate (GTP) gammaS binding), using rat brain synaptosomes. Neither Tamiflu nor GS4071 influenced the re-uptake/release of the 3 monoamines or GTP binding in postsynapses.

The effects of non-medically used psychoactive drugs on monoamine neurotransmission in rat brain.

We developed a reproducible, simple, and small-scale method for determining the re-uptake and release of monoamines (dopamine, serotonin (5-HT) and norepinephrine) using rat brain synaptosomes. These assays were then applied to study the effects of different kinds of non-medically used psychoactive drugs on monoamine re-uptake and release. The phenethylamine derivatives, 4-fluoroamphetamine, 2-methylamino-3,4-methylene-dioxy-propiophenone (methylone), 1-(1,3-benzodioxol-5-yl)-2-butanamine (BDB), and N-methyl-1-(1,3-benzodioxol-5-yl)-2-butanamine (MBDB), had strong inhibitory effects on the re-uptake of dopamine, 5-HT and norepinephrine. 4-Fluoroamphetamine, methylone and BDB also strongly increased the release of the three monoamines, but MBDB increased 5-HT and norepinephrine release, but had little effect on dopamine release. However, 2,5-dimethoxy-4-iodophenethylamine (2C-I), 2,5-dimethoxy-4-ethylphenethylamine (2C-E), 2,5-dimethoxy-4-chlorophenethylamine (2C-C), 2,4,5-trimethoxyamphetamine (TMA-2) and 2,4,6-trimethoxyamphetamine (TMA-6), which are methoxylated phenethylamine derivatives, slightly influenced the re-uptake and release of monoamines. Alpha-metyltryptamine (AMT), a tryptamine derivative, was one of the strongest re-uptake inhibitors and releasers of the three monoamines. The tryptamine derivative, 5-methoxy-alpha-methyltryptamine (5-MeO-AMT), also strongly inhibited re-uptake and increased the release of the three monoamines. N,N-dipropyltryptamine (DPT), 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT), 5-methoxy-N,N-methylisopropyltryptamine (5-MeO-MIPT), and 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) inhibited monoamine re-uptake, but had a few effects on monoamine release. 1-(3-Chlorophenyl)piperazine (3CPP) and 1-(methoxyphenyl)piperazine (4MPP), which are piperazine derivatives, inhibited monoamine re-uptake and accelerated their release. The results suggest that some designer drugs strongly act on the central nerve system to the same extent as restricted drugs.

Effects of N-nitrosofenfluramine, a component of Chinese dietary supplement for weight loss, on CD-1 mice.

Many cases of hepatopathy including deaths have frequently occurred after ingestion of Chinese dietary supplements for weight loss containing N-nitrosofenfluramine (N-fen), a nitroso derivative of fenfluramine (Fen), which was used for the treatment of obesity in the United States. Since Fen decreases appetite by decreasing the serotonin level and exhibits an antibiotic effect, N-fen may have been added, expecting a similar effect. Thus, we synthesized N-fen and orally administered it to mice, and investigated its effect on the liver as well as on the cerebral serotonin nervous system to investigate whether N-fen exhibits an anorectic effect. Three doses of N-fen were orally administered once daily to mice for 1 week. No significant changes in body weight, food intake, and general condition were noted. The liver and kidney weights were significantly increased. On blood chemistry, alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase activities were increased, and total bilirubin and albumin were slightly decreased. On histopathological examination, acidophilic changes and mild cellular swelling were noted in the liver. The liver drug-metabolizing enzyme (P-450) level was significantly higher. The effect of N-fen on the serotonin (5HT) nervous system was examined by quantitative autoradiography of the mouse brain, and it was found that N-fen did not decrease the 5HT nerve activity. Effects of reuptake and release of monoamine neurotransmitters [dopamine (DA), 5HT, and norepinephrine (NE)] were investigated. N-fen inhibited a little 5HT reuptake, and did not inhibit reuptakes of DA and NE. Moreover, N-fen did not affect release of the three monoamines. The above findings suggested that N-fen did not exhibit a serotonin nerve fiber-mediated anorectic effect in mice, but induced hepatopathy.