Species difference of metabolic clearance of bisphenol A using cryopreserved hepatocytes from rats, monkeys and humans.

In vitro metabolism of bisphenol A (BPA), an weak estrogen, was studied with cryopreserved hepatocytes from rat, monkey and human, and was compared with in vivo metabolism reported. The metabolites identified include a major metabolite, BPA glucuronide (BPAG) and BPA sulfate (BPAS). The metabolic rates of bisphenol A at 20micro the hepatocytes (BPAG plus BPAS, nmol/10(6) cells/h) followed the order of rats (48+12)>monkeys (18+4)>humans (8.6+0.8), respectively. The rate of BPAG formation was much higher than that of BPAS formation in all these species. For the BPAG formation, we have determined the apparent K(m) (microf rats (3), monkeys (7), and humans (5). V(max) (nmol/10(6) cells/h) in hepatocytes followed the order of rats (55)>monkeys (22)>humans (11). The total CL(H) for the hepatic formation of BPAG plus BPAS (L/h/kg BW) estimated by well-stirred model with low f(B) value followed the order of rats (3.0)>monkeys (0.68)>humans (0.27), correlating well with in vivo studies of BPA subcutaneously injected rats and monkeys. This study showed that the cryopreserved hepatocytes could be a useful tool for assessing BPA metabolism and predicting systemic exposure of BPA.

Metabolism of acrylamide to glycidamide and their cytotoxicity in isolated rat hepatocytes: protective effects of GSH precursors.

Acrylamide (AA) is a widely studied industrial chemical that is neurotoxic, mutagenic to somatic and germ cells, and carcinogenic in rodents. The recent discovery of AA at ppm levels in a wide variety of commonly consumed foods has energized research efforts worldwide to define toxicity and prevention. Metabolism and cytotoxicity of AA and its epoxide glycidamide (GA) were studied in the hepatocytes freshly isolated from male Sprague-Dawley rats. The isolated hepatocytes metabolized AA to GA. The formation of GA followed Michaelis-Menten kinetic parameters yielded apparent Km = 0.477 +/- 0.100 and 0.263 +/- 0.016 mM, Vmax = 6.5 +/- 2.1 and 26.4 +/- 3.0 nmol/h/10(6) cells, and CLint = 14 +/- 5 and 100 +/- 12 microl/h/10(6) cells for the hepatocytes from untreated and acetone-treated rats, respectively. There were lower Km and marked increases in Vmax (four-fold) and in CLint (sevenfold) in acetone-treated rat hepatocytes. The data suggest that CYP2E1 played a major role in metabolizing AA to more toxic GA. Both AA and GA induced a concentration- and time-dependent glutathione (GSH) depletion of the hepatocytes. From decreasing rates of GSH contents in hepatocytes, the parameters of glutathione S-transferase (GST) in hepatocytes to AA and GA were calculated to be Km = 1.4 and 1.5 mM, Vmax = 21 and 33 nmol/h/10(6) cells, and CLint = 15 and 23 microl/h/10(6) cells, respectively. GA 1.5-times more readily depleted GSH content than AA. GA decreased the viability of hepatocytes at 3 mM, but AA did not. These data indicate that GA is more toxic than AA as assessed by intracellular GSH depletion and loss of viability of hepatocytes. GSH precursors such as N-acetylcysteine and methionine provided significant anti-cytotoxic effects on the decrease of GSH content and cell viability of hepatocytes induced by GA and AA.

Disposition of low doses of 14C-bisphenol A in male, female, pregnant, fetal, and neonatal rats.

Bisphenol A (BPA) is a weak xenestrogen (ADI = 50 microg kg(-1), US EPA) which is mass-produced, with potential for human exposure. To study absorption, distribution, excretion, and metabolism of BPA, BPA labeled with carbon-14 was administered p.o. to male and female Fischer (F344) rats at relatively low doses (20, 100, and 500 microg kg(-1)), and i.v. injected at 100 and 500 microg kg(-1). 14C-BPA (500 microg kg(-1)) was also administered orally to pregnant and lactating rats to examine the transfer of radioactivity to fetuses, neonatal rats, and milk. Radioluminographic determination using phosphor imaging plates was employed to achieve highly sensitive determination of radioactivity. Absorption ratios of radioactivity after three oral doses were high (35-82%); parent 14C-BPA in the circulating blood was quite low, however, suggesting considerable first-pass effect. After an oral dose of 100 microg kg(-1) 14C-BPA, the radioactivity was distributed and eliminated rapidly, but remained in the intestinal contents, liver, and kidney for 72 h. The major metabolite in the plasma and urine was BPA glucuronide, whereas most of the BPA was excreted with the feces as free BPA. A second peak in the time-course of plasma radioactivity suggested enterohepatic recirculation of BPA glucuronide. There was limited distribution of 14C-BPA to the fetus and neonate after oral administration to the dam. Significant radioactivity was not detected in fetuses on gestation days 12 and 15. On day 18, however, radioactivity was detected in the fetal intestine and urinary bladder 24 h after oral dosing of 14C-BPA to the pregnant rats. Part of radioactivity was transferred to neonatal rats from the milk of the treated lactating dam and remained in the intestine of the neonates after 24-h nursing by an untreated dam.

Disposition of a low dose of 14C-bisphenol A in male rats and its main biliary excretion as BPA glucuronide.

Bisphenol A (BPA) is a weak xenoestrogen mass-produced with potential human exposure. The disposition of bisphenol A in male Fischer-344 (F344) rats dosed orally (100 or 0.10 mg/kg) or intravenously (0.10 mg/kg) was determined. Smaller amounts of the dose appeared in the urine. The main excretion route was feces in rats irrespective of dose and administration route. The biliary excretion during 6 h was 58-66% after iv dosing and 45-50% after oral dosing at 0.10 mg 14C-BPA/kg. Toxicokinetic parameters obtained from 14C-BPA-derived radioactivity in blood were the terminal elimination half-life, t1/2beta = 39.5 h, and total body clearance, CLtot = 0.52 l/h/kg after iv dosing of 0.10 mg 14C-BPA/kg to male rats. The blood concentration reached its maximum of 5.5 ng-eq/ml at 0.38 h after oral dose. AUC(0-6 h), AUC(0-48 h), and AUCinf of 14C-BPA-derived radioactivity, were 34, 118, and 192 ng-eqh/ml for the iv dose and 18, 102, and 185 ng-eqh/ml for the oral dose, respectively. The oral bioavailability of F(0-6 h), F(0-48 h), and Finf were 0.54, 0.86, and 0.97, respectively. The 14C-BPA-derived radioactivity was strongly bound to plasma protein (free fraction, fu = 0.046) and preferentially distributed to the plasma with a blood/plasma ratio of 0.67. From the bile of male rats orally dosed at 100 mg/kg, we have isolated and characterized BPA glucuronide (BPA-gluc) by ESI/MS, 1H and 13C NMR spectroscopy. HPLC analysis showed that BPA-gluc was the predominant metabolite in bile and urine. Unchanged BPA was mostly detected in feces. These results suggest that BPA is mainly metabolized to BPA-gluc and excreted into feces through the bile and subject to enterohepatic circulation in rats irrespective of dose and administration route.

Metabolism and excretion of 2-nitro-p-cresol in rats.

Metabolism of 2-nitro- p-cresol (NPC), an important commercial chemical, was studied in female Sprague-Dawley rats, using (14)C-NPC. It was found that NPC was rapidly absorbed and excreted after an oral dose of 250 mg/kg. Approximately 90% of the administered dose was excreted into urine and less than 10% of the dose into feces for 5 days. Urinary and fecal excretion were found to the same extent after 48 h. Bile excretion amounted to approximately 25% for 2 days. Blood levels of (14)C-NPC reached the maximum concentration (39.4 micro g-equivalents/g) within 1 h, and decreased bi-exponentially. The apparent half-lives of (14)C-NPC were 3.8 h for the rapid phase and 37 h for the slow phase, respectively. From studying the distribution in organs at 1.5, 6, 24, 72 and 120 h, we found that the concentrations of radioactivity in various tissues of rats were relatively high in the stomach, intestine, liver, kidney, blood, ovary and uterus. Most organs showed the maximum concentrations at 1.5 h, except for intestine, kidney, ovary, and uterus at 6 h. There was no specific tissue retention after 72 h. Two main conjugate metabolites, glucuronide and sulfate of NPC, were detected with free NPC and 2-acetylamino- p-cresol (AAPC) in the urine. NPC was rapidly absorbed and excreted mainly into urine as the conjugate metabolites. A part of NPC was reduced to 2-amino- p-cresol, followed by acetylation to give AAPC.

Disposition of a low dose of bisphenol a in male and female cynomolgus monkeys.

Bisphenol A (BPA) is a weak estrogenic compound mass-produced with potential human exposure. Following a single oral or intravenous (iv) dose of 100 microg/kg [ring-14C(U)] radiolabeled bisphenol A (14C-BPA) to male and female cynomolgus monkeys, 79-86% of the administered radioactivity was excreted in urine over 7 days, and most of the urinary excretion was recovered by 24 h after dosing, a large part of this occurring within 12 h. The fecal excretion of radioactivity over 7 days was minimal (1.8-3.1%). Toxicokinetic parameters obtained from plasma 14C-BPA-derived radioactivity during 48 h were C(max) = 104-107 ng-eq/ml between 0.25 and 2 h, and AUC(oral) = 244-265 ng-eq*h/ml after oral dosing. In the case of the iv dose, AUC(iv) was 377-382 ng-eq*h/ml, and the bioavailability was 0.66-0.70. The terminal elimination half-life was larger post-iv dose (t(1/2iv) = 13.5-14.7 h) than post-oral dose (t(1/2oral) = 9.63-9.80 h). After iv dose, the fast-phase half-life (t(1/2f)) of total radioactivity was 0.61-0.67 h. The t(1/2f) of unchanged 14C-BPA for females (0.39 h) was smaller than that for males (0.57 h). These results suggested the distribution of lipophilic 14C-BPA in adipose tissue after iv dose, in contrast to first pass metabolism after oral dose. 14C-BPA-derived radioactivity was strongly bound to plasma protein (f(p) = 0.055). Radio-HPLC analysis suggested the predominant plasma and urinary metabolites were mono- and diglucuronide of 14C-BPA and unchanged 14C-BPA was very low (< or =1.5%) after oral dose. These results indicate that the intestinal absorption and metabolism of BPA was rapid and extensive, and the major metabolites, glucuronide conjugates of 14C-BPA, were rapidly excreted into urine in monkeys.