ABSTRACT:Studies have shown that in the rat, bisphenol A (BPA) is metabolized and eliminated primarily as a monoglucuronide, a metabolite without estrogenic activity. The purpose of this study was to determine the extent of monoglucuronide formation in monolayers of hepatocytes from rats, mice, and humans. Noncytotoxic concentrations of BPA (10, 20, and 35 M; 1.0 Ci), as assessed by lactate dehydrogenase leakage, were incubated with isolated hepatocytes for 0-6 h. Media were collected and analyzed for metabolites by radiochemical high performance liquid chromatography and liquid chromatography-tandem mass spectrometry. The metabolites identified include a monoglucuronide (major metabolite), a sulfate conjugate, and a glucuronide/sulfate diconjugate (minor metabolites). In hepatocytes of male Fischer-344 rats, the predominate metabolite was the diconjugate (glucuronide/sulfate). Under these conditions, the extent of metabolism by 3 h was similar in all species tested because all BPA was converted to conjugates by 3 h. Initial rates of metabolism in hepatocytes followed the order of mice > rats > humans. However, when extrapolated to the whole liver (i.e., cells per liver), the hepatic capacity for BPA glucuronidation is predicted to be humans > rats > mice. This research was supported in part by The Society of Plastics Industry Inc., and Southwest Environmental Health Science Center (ES 06694).Bisphenol A (BPA 1 ) is used in the production of polycarbonates, epoxy resins, phenolic resins, and diacrylates. Polycarbonates, one of the most widely used plastics, accounts for 60% of the total production of BPA (Perez et al., 1998). Epoxy-based resins are used in a variety of consumer products that include decorative floor manufacture, lacquer coatings in cans, dental composites and sealants, and as additives in the production of vinyl and acrylic resins. Trace amounts of BPA monomer have been reported to leach out of polycarbonate and epoxy resins (Brotons et al., 1995). Such leaching may result in the potential exposure of humans to trace amounts of BPA.Exposure to BPA is of interest, because it is known to possess weak estrogenic activity (Dodds and Lawson, 1936). In vitro it displaces estradiol from both the ␣ and ␤ estrogen receptor and exhibits weak estrogenic activity (Krishnan et al., 1993, Kuiper et al., 1997. The binding of BPA to the estrogen receptor has been reported to be 10,000 times less than that of 17␤-estradiol (Gaido et al., 1997). Following oral exposure, BPA undergoes its first pass metabolism in the intestine and/or liver, which greatly limits its systemic bioavailability (Pottenger et al., 2000;Upmeier et al., 2000). In vitro data show that BPA is rapidly conjugated with glucuronic acid by hepatic rat microsomes (Yokota et al., 1999). Recent results obtained using rat hepatocytes (HC) or perfused liver confirm the extensive formation of a BPA monoglucuronide (Nakagawa and Tayama, 2000;Inoue et al., 2001) and correlate with in vivo findings that demonstrated the BPA-glucuronide to be the major me...

Substrate derived biomarkers are necessary in slowly progressing monogenetic diseases caused by single enzyme deficiencies to identify affected patients and serve as surrogate markers for therapy response. N-glycanase 1 (NGLY1) deficiency is an ultra-rare autosomal recessive disorder characterized by developmental delay, peripheral neuropathy, elevated liver transaminases, hyperkinetic movement disorder, and (hypo)-alacrima. We demonstrate that N-acetylglucosamine-asparagine (GlcNAc-Asn; GNA), is the analyte most closely associated with NGLY1 deficiency, showing consistent separation in levels between patients and controls. GNA accumulation is directly linked to the absence of functional NGLY1, presenting strong potential for its use as a biomarker. In agreement, a quantitative LC-MS/MS assay, developed to assess GNA from 3 to 3000 ng/mL, showed it is conserved as a marker for loss of NGLY1 function in NGLY1 deficient cell lines, rodents (urine, cerebrospinal fluid, plasma, and tissues), and patients (plasma and urine). Elevated GNA levels differentiate patients from controls, are stable over time, and correlate with changes in NGLY1 activity. GNA as a biomarker has the potential to identify and validate patients with NGLY1 deficiency, act as a direct pharmacodynamic marker, and serve as a potential surrogate endpoint in clinical trials.