Recently, disposable gloves made of polyvinyl chloride (PVC) containing di(2-ethylhexyl)phthalate (DEHP) were prohibited for food-contact uses such as cooking and serving because high DEHP levels were detected in packed lunches for sale at convenience stores and patients' meals in hospitals.1,2) Endocrine-disrupting effects are suspected for phthalate diesters, including DEHP, that are widely used as plasticizers. Endocrine disruption is thought to occur by in vivo metabolism to phthalic acid mono esters.3) Accordingly, disposable PVC gloves were replaced by those composed of various materials such as polyethylene, natural rubber, PVC not containing DEHP, and nitrile-butadiene rubber (NBR). Until recently, NBR gloves were rarely used in the food industry in Japan. However, as a result of the prohibition of PVC gloves, NBR gloves have been increasingly used for preparation of food. Mutsuga et al. 4,5) isolated and identified chemicals eluted from NBR gloves, including 4,4Ј-butylidenebis(6-t-butyl-m-cresol) (BBBC), 2,4-di-t-butylphenol (DBP), and 2,2,4-trimetyl-1,3-pentanediol diisobutyrate (TPDB).We have examined the hormonal effects of many chemicals (parabens, alkylphenols, styrene oligomers, etc.) using in vitro and in vivo assays. [6][7][8][9][10][11][12][13] For example, styrene oligomers, especially styrene trimers, are contaminants in polystyrene that is widely used for food containers. We reported that the styrene trimer 1e-phenyl-4a-(1Ј-phenyethyl)-tetralin (ST-4) had antiandrogenic-like and estrogenic-like activity in vitro, 7,14) and the effect of ST-4 on the offspring of pregnant rats were further examined.13) The relative anogenital distance was significantly shortened, and the relative weights of the brain, prostate, testis and epididymides, as well as the Sertoli cell counts were significantly decreased. Serum follicle stimulating hormone and luteinizing hormone level were remarkably reduced, but the testosterone level was increased. In this report, we studied the hormonal effects of BBBC, DBP, and TPDB eluted from NBR gloves, as well as the effects of 4,4Ј-thiobis(6-t-butyl-m-cresol) (TBBC), which is similarly used as a plasticizer, and 2,2Ј-methylenebis(4-ethyl-6-t-butylphenol) (MBBP) and 2,2Ј-methylenebis(6-tbutyl-p-cresol) (MBBC), which are the structurally homologous to BBBC. We used several types of in vitro assays (AREcoScreen reporter gene assay, MVLN cell reporter gene assay, competitive binding assays for the androgen receptor (AR) and estrogen receptor (ER)).
MATERIALS AND METHODSReagents Test chemicals were the highest grade available for sale or environmental analysis. The source, purity, CAS No., and abbreviation of chemicals are listed in Table 1. The structures of the chemicals are indicated in Fig. 1. Test chemicals were dissolved in dimethyl sulfoxide (DMSO) for AR-and ER-luciferase reporter gene assays and AR-and ERa-competitive binding assays, and the final concentration of DMSO in the culture and binding assay medium was adjusted to 0.1%. Testosterone and bisphenol A (BPA) were...