Ryo Kamata

Screening and detection of the in vitro agonistic activity of xenobiotics on the retinoic acid receptor

The retinoic acid receptors (RARs) play key roles in various biological processes in response to endogenous retinoic acids. However, excessive embryonic exposure to specific ligands for each subtype of the RAR was reported to induce specific developmental abnormalities. We measured the RAR agonistic activity of 543 chemicals using an assay system adopting yeast cells transfected with the human RAR gamma and a coactivator. Eighty-five of the 543 chemicals, including 16 organochlorine pesticides, 14 styrene dimers, 9 monoalkylphenols and 6 parabens, exhibited RAR gamma agonistic effects in this assay. In particular, monoalkylphenols having a 6-9 carbon alkyl group para to the phenolic hydroxyl group possessed high affinity for the RAR gamma, and their activities were 1.363-0.446% of that of all-trans RA. para-Alkylphenols chlorinated at the ortho position also were about as active or more active than their unchlorinated analogs. In addition, all tested styrene dimers showed positive effects, and the activity of 1-phenyltetralin, the strongest in this category, was 1.169% that of all-trans RA. A number of chemicals having binding affinity for the RAR gamma were revealed in this study (both newly identified and confirmed), further comprehensive studies of in vitro and in vivo effects via the RARs are required for the reliable risk assessment of chemicals. In vitro receptor binding studies represent an important step in hazard identification and suggest a potential mechanism of action, which can be an important step in risk assessment and in particular for screening studies to identify potential toxicity and inform mechanistic studies.

Evaluation of estrogenic activity of parabens and their chlorinated derivatives by using the yeast two‐hybrid assay and the enzyme‐linked immunosorbent assay

We assessed the estrogen agonist activities of 21 parabens and their chlorinated derivatives by using yeast two-hybrid assays incorporating either the human or medaka (Oryzias latipes) estrogen receptor alpha (hERalpha and medERalpha, respectively), and by using hERalpha competitive enzyme-linked immunosorbent assay (ER-ELISA). In the two-hybrid assay with hERalpha, five parabens and three chlorinated derivatives exhibited estrogenic activity, and their relative activity (17beta-estradiol [E2] = 1) ranged from 2.0 x 10(-5) to 2.0 x 10(-4), with the highest activity observed in i-butylparaben. In the medERalpha assay, six parabens and six chlorinated derivatives exhibited estrogenic activity and their relative activity ranged from 2.7 x 10(-5) to 3.5 x 10(-3), with the highest activity observed in benzylparaben, its monochlorinated derivative, i-butylparaben, and n-butylparaben. Although medERalpha demonstrated an activity to E2 that was three times lower than that demonstrated by hERalpha, medERalpha has a higher sensitivity to parabens than hERa (1.3-8.9 times). Five parabens and two chlorinated derivatives exhibited a binding affinity to ERa in the ER-ELISA; of the parabens, i-butylparaben exhibited the strongest binding affinity. The yeast two-hybrid assay and the ER-ELISA also revealed that many of the assayed chlorinated parabens were much weaker than the parent compound. In addition, the results mainly showed that parabens with a bulk substituent (e.g., i-butyl and benzyl groups) had a higher activity than those with a sterically small substituent. It is considered that derivatization masks the apparent estrogenic activity of parabens, but the resulting chlorinated compounds may represent a potential hazard and therefore other toxicity tests should be performed to determine the toxicity of the chlorinated derivatives.

A pilot survey of 39 Victorian WWTP effluents using a high speed luminescent umu test in conjunction with a novel GC-MS-database technique for automatic identification of micropollutants

In 2007, samples of treated effluent were collected at point of discharge to the environment from 39 wastewater treatment plants (WWTPs) located across Victoria, Australia grouped by treatment type. Sample genotoxicity was assessed with a high-throughput luminescent umu test method using Salmonella typhimurium TL210 strain, with and without addition of a commercially available metabolic activation system. Samples were also screened using a gas chromatographic-mass spectrometric mass-structure database recognition method. A genotoxic response was observed in half of the samples tested without metabolic activation system (<LOR - 0.19 μg/L 4-nitroquinoline-N-oxide equivalents). On addition of metabolic activation system, 75% of samples elicited a genotoxic response, the majority of responses were stronger than without metabolic activation (<LOR - 2.97 μg/L benzo[a]pyrene equivalents). The type of WWTP had no effect on genotoxicity. A large number of chemicals were identified in the effluents, although none could be unambiguously tied to the genotoxicity observed. Chemicals observed in one or more effluents included food additives (e.g. dibenzylether), various alkyl phenols, tyre leachates (e.g. 2(3H)-benzothiazolone), antioxidants, flame retardants (e.g. tris(2-chloroethyl)phosphate), insect repellents (e.g. diethyltoluamide), stimulants (e.g. caffeine) and anticonvulsants (e.g. carbamazepine). Of the 451 pesticides screened, carbamate insecticides (e.g. bendiocarb, propoxur), plant growth regulators (e.g. propham) and herbicides (e.g. atrazine, metolachlor, simazine) were amongst the compounds observed.

Reevaluation of the developmental toxicity of dieldrin by the use of fertilized Japanese quail eggs

To reevaluate the toxicity of the organochlorine insecticide and persistent organic pollutant dieldrin and confirm its impact on development, an exposure trial using bird eggs was performed. Dieldrin at concentrations of 10-100 microg/g of egg was injected into the yolks of Japanese quail (Coturnix japonica) eggs. Hatchlings from the eggs were raised to sexual maturity and multiple tests to detect the harmful effects of dieldrin were conducted. Dieldrin at 100 microg/g decreased egg hatchability by 50.0% (vehicle control, 86.7%), although embryogenesis even in unhatched eggs treated with high doses of dieldrin was normal. In safely hatched chicks, dose-dependent early death with tonic seizure was observed and all birds exposed to 100 microg/g died within 3 days. Other significant alterations in hatchlings were enlargement of the whole brain, decreases in mRNA expressions of tryptophan hydroxylase in the brainstem and cholesterol side-chain cleavage in the male gonad, and increases in mRNA expressions of cytochrome P450 1A and 2C18 in the liver. For mature birds (males at 5 weeks and females at 10 weeks of age), impairment of eggshell formation such as reduced eggshell mass and eggshell thinning, increases in the body mass of males and the liver mass of females and increases in serum total cholesterol and triglyceride concentrations were observed. The results indicated that not only does the neurotoxicity of dieldrin bring early death, but its effects on reproductive and hepatic functions (detected as gene transcriptional changes in hatchlings) persist harmfully after maturity.

The feasibility of using mosquitofish (Gambusia affinis) for detecting endocrine‐disrupting chemicals in the freshwater environment

We evaluated the utility of gene-transcriptional responses in the liver of mosquitofish (Gambusia affinis), a species introduced to many countries and therefore widely available, for detecting endocrine-disrupting activity in water. Exposure to β-naphthoflavone, an aryl hydrocarbon receptor (AhR) agonist, significantly increased the transcript of the cytochrome P4501A gene (cyp1a), peaking at 24 h, in both sexes at concentrations of 10 µg/L or more. 17β-Estradiol (E(2) ) at 500 ng/L increased the number of males showing gene transcription of precursors of yolk protein, vitellogenin (Vtga, Vtgb, and Vtgc), at 24, 48, and 72 h. Exposure for 48 h to bisphenol A (BPA), an estrogen mimic, also increased vtg-positive males at 1 mg/L or more. Leachate from a Japanese stable-type landfill significantly increased vtg-positive males after 48 h exposure, and the in vitro activity of the leachate against the estrogen receptor (ER) was estimated as an E(2) equivalent of 240 ng/L by yeast transfected with the ER. Chemical analysis showed that major contributors to the ER activation were BPA and 4-tert-octylphenol. This leachate and drainage water from a control-type landfill had AhR activities, estimated by yeast with the AhR, but had no significant effect on cyp1a transcription. These results showed that mosquitofish are suitable for detecting in vivo AhR and ER effects, but are insensitive to E(2).