No abstract

Phthalate esters are commonly used plasticizers; however, some are suspected to cause reproductive toxicity. Administration of high doses of di-(2-ethylhexyl) phthalate (DEHP) induces germ cell death in male rodents. Mono-(2-ethylhexyl) phthalate (MEHP), a hydrolyzed metabolite of DEHP, appears to be responsible for this testicular toxicity; however, the underlying mechanism of this chemical's action remains unknown. Here, using a one-step affinity purification procedure, we identified glycogen debranching enzyme (GDE) as a phthalate-binding protein. GDE has oligo-1,4-1,4-glucanotransferase and amylo-1,6-glucosidase activities, which are responsible for the complete degradation of glycogen to glucose. Our findings demonstrate that MEHP inhibits the activity of oligo-1,4-1,4-glucanotransferase, but not of amylo-1,6-glucosidase. Among various phthalate esters tested, MEHP specifically binds to and inhibits GDE. We also show that DEHP administration affects glycogen metabolism in rat testis. Thus, inhibition of GDE by MEHP may play a role in germ cell apoptosis in the testis.

Bisphenol A (BPA) forms the backbone of plastics and epoxy resins used to produce packaging for various foods and beverages. BPA is also an estrogenic disruptor, interacting with human estrogen receptors (ER) and other related nuclear receptors. Nevertheless, the effects of BPA on human health remain unclear. The present study identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a novel BPA-binding protein. DNA-PKcs, in association with the Ku heterodimer (Ku70/80), is a critical enzyme involved in the repair of DNA double-strand breaks. Low levels of DNA-PK activity are previously reported to be associated with an increased risk of certain types of cancer. Although the Kd for the interaction between BPA and a drug-binding mutant of DNA-PKcs was comparatively low (137 nM), high doses of BPA were required before cellular effects were observed (100–300 μM). The results of an in vitro kinase assay showed that BPA inhibited DNA-PK kinase activity in a concentration-dependent manner. In M059K cells, BPA inhibited the phosphorylation of DNA-PKcs at Ser2056 and H2AX at Ser139 in response to ionizing radiation (IR)-irradiation. BPA also disrupted DNA-PKcs binding to Ku70/80 and increased the radiosensitivity of M059K cells, but not M059J cells (which are DNA-PKcs-deficient). Taken together, these results provide new evidence of the effects of BPA on DNA repair in mammalian cells, which are mediated via inhibition of DNA-PK activity. This study may warrant the consideration of the possible carcinogenic effects of high doses of BPA, which are mediated through its action on DNA-PK.

Mammalian pyruvate dehydrogenase complex (PDHC) is a multienzyme complex with a total Mr of approximately 7 × 106. It consists of five components: pyruvate dehydrogenase (El; EC 1.2.4.1), dihydrolipoyl transacetylase (E2; EC 2.3.1.12), lipoamide dehydrogenase (E3; EC 1.6.4.3) and two specific regulatory enzymes. E1 consists of two different peptides, EI~ and EI~. An additional component of PDHC, protein X, has been elucidated immunochemically.PDHC deficiency is a rare disease and is a cause of primary lactic acidosis. While there are many reports of this disease, there are few analyses of its molecular basis in the literature. We report here our immunochemical analyses of PDHC in skin fibroblasts and/or Epstein-Barr transformed lymphoid cells obtained from three patients with this disease. MATERIALS AND METHODSCase 1: The proband was a 1-year-old girl delivered by cesarian section at a full term. Ventricular dilatation in the fetus was detected by ultrasound. At birth, there was a slight cyanosis, her cry and muscle tonus were weak and her fingers were contracted. Brain CT scan showed ventricular dilatation and agenesis of the corpus callosum. Serum lactate and pyruvate were greatly elevated, the values being lactate 112.9mg/dl and pyruvate 8.6mg/dl.Case 2: This patient was a 9-year-old girl born after a full-term pregnancy and spontaneous delivery. Tube feeding was required because of a poor sucking ability from birth on. Delay in development was prominent at the age of 3 months when she had no visual response or head control. At 6 months, she was prescribed anticonvulsant drug therapy for infantile myoclonic seizures which had become difficult to control. Brain CT scan showed a diffuse brain atrophy. The levels of lactate (41.2mg/dl) and pyruvate (3.9mg/dl) were elevated.Case 3: This patient was a 14-year-old boy. He was born after a normal delivery and term pregnancy and he developed normally. At age 3 years, his mother noticed 329Journal of Inherited Metabolic Disease. ISSN 0141-8955. Coovright (~ SSIEM and MTP Pres~ I .irnlted