Influence of Matrigel-overlay on constitutive and inducible expression of nine genes encoding drug-metabolizing enzymes in primary human hepatocytes.

1. Previous studies reported that rat hepatocytes overlaid with extracellular matrix components (Matrigel) maintain the expression and responsiveness of drug-metabolizing enzymes. However, whether Matrigel provides similar advantages in human hepatocytes remains largely uncertain.2. The influence of Matrigel-overlay on the constitutive and phenobarbital- and oltipraz-inducible expression of nine biotransformation enzymes, cytochrome P450s 1A1, 1A2, 2B6, 3A4, and glutathione S-transferases A1, A2, M1, T1, P1, in primary human hepatocytes was evaluated.3. Hepatocytes from five livers were maintained on a rigid collagen substratum with or without Matrigel overlay and treated for 48?h with two doses of each inducer. Quantitative RT-PCR, and for selected genes, immunoblot and enzyme activity analyses, demonstrated that human hepatocytes overlaid with Matrigel showed consistently higher constitutive and inducible expression of biotransformation genes. 4. Phenobarbital-mediated responsiveness of cytochrome P450 2B6, a potential indicator of hepatocyte differentiation status, was markedly higher in overlaid relative to non-overlaid hepatocytes. 5. It is concluded that an Matrigel overlay facilitates the maintenance and induction of xenobiotic metabolizing enzymes in primary cultures of human hepatocytes.

Phytochemical-induced changes in gene expression of carcinogen-metabolizing enzymes in cultured human primary hepatocytes.

1. The naturally occurring compounds curcumin (CUR), 3,3'-diindolylmethane (DIM), isoxanthohumol (IXN), 8-prenylnaringenin (8PN), phenethyl isothiocyanate (PEITC) and sulforaphane (SFN) protect animals against chemically induced tumours. Putative chemoprotective mechanisms include modulated expression of hepatic biotransformation enzymes. However, few, if any, studies have used human primary cells as test models. 2. The present study investigated the effects of these phytochemicals on the expression of four carcinogenesis-relevant enzymes--cytochrome P450 (CYP)1A1 and 1A2, NAD(P)H:quinone oxidoreductase (NQO1) and glutathione S-transferase A1 (GSTA1)--in primary cultures of freshly isolated human hepatocytes. 3. Quantitative RT-PCR analyses demonstrated that CYP1A1 was up-regulated by PEITC and DIM in a dose-dependent manner. CYP1A2 transcription was significantly activated following DIM, IXN, 8PN and PEITC treatments. DIM exhibited a remarkably effective induction response of CYP1A1 (474-, 239- and 87-fold at 50, 25 and 10 microM, respectively) and CYP1A2 (113-, 70- and 31-fold at 50, 25 and 10 microM, respectively), that was semiquantitatively reflected in protein levels. NQO1 expression responded to PEITC (11 x at 25 microM), DIM (4.5 x at 50 microM) and SFN (5 x at 10 microM) treatments. No significant effects on GSTA1 transcription were seen. 4. The findings show novel and unexpected effects of these phytochemicals on the expression of human hepatic biotransformation enzymes that play key roles in chemical-induced carcinogenesis.

The kinetics of aflatoxin B1 oxidation by human cDNA-expressed and human liver microsomal cytochromes P450 1A2 and 3A4.

The combined presence of CYP1A2 and 3A4, both of which oxidize aflatoxin B1 (AFB1) to the reactive aflatoxin B1-8,9-epoxide (AFBO) and to hydroxylated inactivation products aflatoxin M1 (AFM1) and aflatoxin Q1 (AFQ1), substantially complicates the kinetic analysis of AFB1 oxidation in human liver microsomes. In the present study, we examine the reaction kinetics of AFB1 oxidation in human liver microsomes (HLMs, N = 3) and in human CYP3A4 and CYP1A2 cDNA-expressed lymphoblastoid microsomes for the purpose of identifying the CYP isoform(s) responsible for AFB1 oxidation at low substrate concentrations approaching those potentially encountered in the diet. AFBO formation by cDNA-expressed human CYP1A2 followed Michaelis-Menten kinetics (Km = 41 microM, Vmax = 2.63 nmol/min/nmol P450). Furthermore, the portion of AFBO formed in HLMs which was eliminated by furafylline, a specific mechanism-based inhibitor of CYP1A2, also followed Michaelis-Menten kinetics (Km = 32-47 microM, Vmax = 0.36-0.69 nmol/min/nmol P450). The formation of AFBO (activation product) and AFQ1 (detoxification product) in cDNA-expressed human CYP3A4 microsomes was sigmoidal and consistent with the kinetics of substrate activation. Accordingly, application of a sigmoid Vmax model equivalent to the Hill equation produced excellent fits to the cDNA-expressed CYP3A4 data and also to the data from HLMs pretreated with furafylline to remove CYP1A2. The Hill model predicted that two substrate binding sites are involved in CYP3A4-mediated AFB1 catalysis and that the average affinity of AFB1 for the two sites was 140-180 microM. Vmax values for AFQ1 formation were 10-fold greater than those for AFBO, and total substrate turnover to both was 67 nmol/min/nmol CYP3A4. Using the derived kinetic parameters for CYP1A2 and 3A4 to model the in vitro rates of AFB activation at low substrate concentrations, it was predicted that CYP1A2 contributes to over 95% of AFB activation in human liver microsomes at 0.1 microM AFB. The important role of CYP1A2 in the in vitro activation of AFB at low substrate concentrations was supported by DNA binding studies. AFB1-DNA binding in control HLMs (reflecting the contribution of CYP1A2 and CYP3A4) and furafylline-pretreated microsomes (reflecting the contribution of CYP3A4 only) catalyzed the binding of 1.71 and 0.085 pmol equivalents of AFB1 to DNA, respectively, indicating that CYP1A2 was responsible for 95% of AFB1-DNA adduct formation at 0.133 microM AFB. These results demonstrate that CYP1A2 dominates the activation of AFB in human liver microsomes in vitro at submicromolar concentrations and support the hypothesis that CYP1A2 is the predominant enzyme responsible for AFBO activation in human liver in vivo at the relatively low dietary concentrations encountered in the human diet, even in high AFB exposure regions of the world. However, because the actual concentrations of AFB in liver in vivo following dietary exposures are uncertain, additional studies in exposed human populations are needed. Quantitative data on the relative rates of AFM1 and AFQ1 excretion (potential biomarkers for CYP1A2 and 3A4 activity, respectively) in humans would be useful to validate the actual contributions of these two enzymes to AFB1 oxidation in vivo.

The effects of diquat and ciprofibrate on mRNA expression and catalytic activities of hepatic xenobiotic metabolizing and antioxidant enzymes in rat liver.

Although the mechanisms responsible for chemically induced oxidative stress are under intense investigation, little is known about the effects of prooxidant chemicals on the expression of drug-metabolizing enzymes. We examined the effects of diquat (0.1 mmol/kg, ip) and ciprofibrate (0.025% w/w, diet), chemicals which induce oxidative stress via different biochemical mechanisms, on the steady-state messenger RNA (mRNA) levels of six cytochrome P450 enzymes, seven glutathione S-transferase (GST) isoenzymes, UDP-glucuronosyl transferase 1-06 (UGT1*06), gamma-glutamylcysteine synthetase (gamma GCS), NADP(H):quinone oxidoreductase (quinone reductase), Cu/Zn superoxide dismutase (SOD), catalase, and 18S ribosomal RNA in the livers of male Sprague-Dawley rats. Effects of chemical treatments on mRNA levels were compared to changes in catalytic activities for selected enzymes. Ciprofibrate treatment selectively decreased CYP1A2 mRNA expression, whereas both chemicals suppressed CYP3A2 mRNA expression. CYP4A1 mRNA expression and lauric acid hydroxylase activities were induced by ciprofibrate treatment, whereas diquat treatment moderately increased CYP4A1 mRNA levels without affecting lauric acid hydroxylase activities. The steady-state mRNA levels encoding constitutively expressed GST isozymes (Ya1, Ya2, Yb1, Yb2, and Yc1) were decreased by diquat exposure, and the mRNA encoding four of the five constitutively expressed GSTs (Ya1, Ya2, Yb1, and Yc1) were also decreased by ciprofibrate treatment. Nonconstitutively expressed or low constitutively expressed genes (CYP1A1, CYP2B1, CYP2B2, GST Yc2, GST Yf, and UGT1*06) were not induced by exposure to the prooxidants. Changes in isozyme-specific catalytic activities were more consistent with the observed changes in mRNA expression for the GSTs than for the P450s. Both treatments had inhibitory effects on hepatic GSH biosynthesis by decreasing gamma GCS large-subunit mRNA expression, gamma GCS catalytic activities, and hepatic GSH concentrations. Cu/Zn SOD and quinone reductase mRNA levels were increased after ciprofibrate exposure, whereas Cu/Zn SOD mRNA expression was decreased in the diquat-treated animals. The results of this study indicate that diquat and ciprofibrate can decrease the expression profile of a number of phase I, phase II, and antioxidant enzymes and inhibit GSH biosynthesis. These effects may involve the pretranslational loss of hepatic mRNAs, possibly due to accelerated production of reactive oxygen species.

Human variability in hepatic glutathione S-transferase-mediated conjugation of aflatoxin B1-epoxide and other substrates.

Hepatic cytosolic fractions prepared from 14 human donors were analysed for glutathione S-transferase (GST) activity towards synthetic aflatoxin B1-8,9-epoxide (AFBO). In addition, GST-AFBO activity of pooled human liver cytosols was compared with rat, hamster, and mouse liver cytosol GST-AFBO activities. Consistent with previous studies, human liver cytosolic GSTs exhibited little activity towards AFBO. Hepatic GST-AFBO activities of rat, hamster, and mouse were 48-, 56-, and 312-fold greater, respectively, than observed for human liver using synthetic AFBO, and 70-, 465-, and 3545-fold greater, respectively, than observed for human liver using microsomally-generated AFBO. Furthermore, there was a 58-fold variation in hepatic GST-AFBO activities among the 14 human samples using synthetic AFBO as a substrate. Large interindividual variations were also observed with respect to GST activities towards bromosulfophthalein (BSP, 92-fold variation) and 3,4-dichloronitrobenzene (DCNB, 36-fold variation). Lesser interindividual variations were observed with respect to human liver GST activities towards benzo(a)pyrene-4,5-oxide (BaPO, 9-fold variation), 1-chloro-2,4-dinitrobenzene (CDNB, 8.5-fold variation), cumene hydroperoxide (CHP, 5-fold variation), and p-nitrophenyl acetate (NPA, 4-fold variation). No correlation was found among GST-AFBO activities and the presence of GST mu as determined by enzyme-linked immunosorbent assay (ELISA) or GST-trans-stilbene oxide (TSO) catalytic activity. Our observations support those of previous studies indicating that human liver cytosolic GSTs are relatively ineffective at conjugating AFBO. Furthermore, our data indicate that humans exhibit large inter-individual differences with respect to hepatic cytosolic GST conjugation of AFBO and certain other GST substrates.

Role of human microsomal and human complementary DNA-expressed cytochromes P4501A2 and P4503A4 in the bioactivation of aflatoxin B1.

The metabolism of the carcinogenic mycotoxin aflatoxin B1 (AFB1) was examined in microsomes derived from human lymphoblastoid cell lines expressing transfected CYP1A2 or CYP3A4 complementary DNAs and in microsomes prepared from human liver donors (n = 4). Lymphoblast microsomes expressing only CYP1A2 activated AFB1 to AFB1-8,9-epoxide (AFB1-8,9-epoxide trapped as the glutathione, conjugate) at both 16 microM and 128 microM AFB1 concentrations, whereas activation of AFB1 to the epoxide in lymphoblast microsomes expressing only CYP3A4 was detected only at high substrate concentrations (128 microM AFB1). AFB1 epoxidation was strongly inhibited in CYP1A2 but not CYP3A4 lymphoblast microsomes pretreated with furafylline, a specific mechanism-based CYP1A2 inhibitor, whereas troleandomycin (TAO), a specific CYP3A inhibitor, strongly inhibited AFB1 epoxidation in CYP3A4 but not CYP1A2 microsomes. Formation of the hydroxylated metabolite aflatoxin M1 (AFM1) was observed only in the CYP1A2 microsomes whereas aflatoxin Q1 (AFQ1) production was observed exclusively in the CYP3A4 microsomes. Treatment of individual human liver microsomes (HLM) with TAO resulted in an average 20% inhibition of AFB1-8,9-epoxide formation at 16 microM AFB1, whereas incubation of HLM with furafylline at 16 microM AFB1 resulted in an average 72% inhibition of AFB1-8,9-epoxide formation at 16 microM AFB1. TAO was slightly more effective than furafylline in inhibiting AFB1 epoxidation at 128 microM AFB1 (46% inhibition by TAO, 32% inhibition by furafylline) in HLM. AFB1-8,9-epoxide formation was inhibited by 89% at low substrate concentration and 85% at high substrate concentrations when HLM were inhibited with a furafylline/TAO mixture. AFM1 formation was strongly inhibited by furafylline, whereas AFQ1 formation was strongly inhibited by TAO, in all HLM regardless of substrate concentration. Analysis of R-6- and R-10-hydroxywarfarin activities (respective markers of CYP1A2 and CYP3A4 activities) in the complementary DNA-expressed microsomes demonstrated that TAO was less effective than furafylline as a selective P450 isoenzyme inhibitor (60% inhibition of CYP3A4 by TAO as compared to 99% inhibition of CYP1A2 by furafylline). The rates of AFB1 epoxidation and AFQ1 formation in HLM were increased 7- and 18-fold, respectively, at high versus low substrate concentrations. These results are consistent with the hypothesis that CYP1A2 is the high-affinity P450 enzyme principally responsible for the bioactivation of AFB1 at low substrate concentrations associated with dietary exposure. CYP3A4 appears to have a relatively low affinity for AFB1 epoxidation and is primarily involved in AFB1 detoxification through AFQ1 formation in HLM.(ABSTRACT TRUNCATED AT 400 WORDS)

Neonatal and infant heart transplantation.

Since the advent of cyclosporine A, cardiac transplantation has been offered routinely for adolescents and children with cardiomyopathy refractory to medical management. Recently, cardiac transplantation has been offered to neonates with severe structural congenital heart disease. This article reviews the indications, pretransplant evaluation, and post-transplant management of neonates and infants undergoing cardiac transplantation.

Simultaneous determination of cytosolic glutathione S-transferase and microsomal epoxide hydrolase activity toward benzo[a]pyrene-4,5-oxide by high-performance liquid chromatography.

Cytosolic glutathione S-transferase (GST) and microsomal epoxide hydrolase (EH) are important detoxification enzymes for many epoxide xenobiotics. We have developed a rapid, simple, and convenient HPLC assay which measures both of these enzyme activities toward benzo[a]pyrene-4,5-oxide (BaPO) in tissue homogenates. Tissue fractions were incubated at 37 degrees C in the presence of 5 mM glutathione. Reactions were initiated by addition of BaPO and terminated by the addition of ice-cold acetonitrile containing 2-methoxynaphthalene as an internal standard. Samples were analyzed directly on a 15-cm C18 reverse-phase column at room temperature, with a ternary solvent program which utilized 0.01% ammonium phosphate buffer (pH 3.5), acetonitrile, and water. The uv absorbance (260 nm) was monitored. Baseline resolution of BaPO, BaPO-GSH, and BaPO-diol and the internal standard was accomplished in 10 min. In rat hepatic S9, production of both BaPO-GSH and BaPO-diol was linear with time and protein up to 15 min and 500 micrograms/ml, respectively. Coefficients of variation for replicate analyses were 2.7 and 3.7% for GST and EH activities in S9, respectively. With fluorescence detection (ex, 241; em, 389 nm), this assay was sensitive enough to measure GST and EH activities in mononuclear leukocytes (MNL). GST and EH activities in 109 human MNL samples were 142 +/- 74 (mean +/- SD; range 21-435) pmol/mg/min and 19 +/- 9 (mean +/- SD; range 3-59) pmol/mg/min, respectively. These results demonstrate the simplicity, high sensitivity, and applicability of this assay for a broad range of tissues.

Hypochlorous acid-activated carbon: an oxidizing agent capable of producing hydroxylated polychlorinated biphenyls.

Granular activated carbon (GAC), in the presence of dilute aqueous hypochlorite solutions typical of those used in water treatment, was converted to a reagent capable of carrying out free-radical coupling reactions and other oxidations of dilute aqueous solutions of phenols. The products included biphenyls with chlorine and hydroxyl substitution (hydroxylated polychlorinated biphenyls). For example, 2,4-dichlorophenol, a common constituent of wastewaters and also natural waters treated with hypochlorite, was converted to 3,5,5'trichloro-2,4'-dihydroxybiphenyl and several related compounds in significant amounts. It is possible that these products pose more of a health hazard than either the starting phenols or the unhydroxylated polychlorinated biphenyl derivatives.