Biomedical subjects

D L Eaton

Publications and source records attributed to D L Eaton.

At least 55 records · Page 3Linked to original sources

PubMed1996

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.

Adult↗

PubMed1996

Assessment of regional cytochrome P450 activities in rat liver slices using resorufin substrates and fluorescence confocal laser cytometry.

Characterizing constitutive activities and inducibility of various cytochrome P450 isozymes is important for elucidating species and individual differences in susceptibility to many toxicants. Although expression of certain P450s has been studied in homogenized tissues, the ability to assess functional enzyme activity without tissue disruption would further our understanding of interactive factors that modulate P450 activities. We used precision-cut, viable rat liver slices and confocal laser cytometry to determine the regional enzyme activities of P450 isozymes in situ. Livers from control and beta-naphthoflavone (beta NF)-treated rats were sectioned with a Krumdieck tissue slicer into 250-microns thick sections. A slice perfusion chamber that mounts on the cytometer stage was developed to allow for successive measurement of region-specific P450-dependent O-dealkylation of 7-ethoxy-, 7-pentoxy-, and 7-benzyloxyresorufin (EROD, PROD, and BROD activity, respectively) in the same liver slice. Images of the accumulated fluorescent resorufin product within the tissue were acquired using a confocal laser cytometer in confocal mode. As expected, slices isolated from beta NF-treated rats showed high levels of centrilobular EROD activity compared to slices from control rats, whereas PROD and BROD activities remained at control levels. These techniques should allow for the accurate quantification of regional and cell-specific P450 enzyme activity and, with subsequent analysis of the same slice, the ability to correlate specific P450 mRNAs or other factors with enzymatic activity. Moreover, these techniques should be amenable to examination of similar phenomena in other tissues such as lung and kidney, where marked heterogeneity in cellular P450 expression patterns is also known to occur.

Analysis of Variance↗

PubMed1995

Activation and characterization of procarboxypeptidase B from human plasma.

Recently we reported the isolation and cloning of a novel plasma procarboxypeptidase B that binds plasminogen [Eaton, D. L., Malloy, B. E., Tsai, S. P., Henzel, W., & Drayna, D. (1991) J. Biol. Chem. 266, 21833-21838]. This plasma procarboxypeptidase is structurally similar to tissue procarboxypeptidases, and initial substrate studies showed that this plasma protein behaves like a basic carboxypeptidase and is now known as human plasma procarboxypeptidase B (pro-pCPB). However, unlike the tissue procarboxypeptidases, pro-pCPB is extremely unstable to trypsin activation. Trypsin cleaves pro-pCPB at two sites: Arg-92 and Arg-330. Cleavage at Arg-92 releases the activation peptide and generates an active enzyme. However, cleavage at Arg-330 inactivates pCPB. This renders the characterization of pCPB difficult. We have found that 6-amino-n-hexanoic acid (epsilon ACA), a compeptitive inhibitor of basic carboxypeptidases, selectively limits trypsin cleavage of pro-pCPB. In the presence of epsilon ACA, trypsin cleavage at Arg-330 is significantly limited while the cleavage at Arg-92 is unaffected. Using this approach, active pCPB can now be obtained. Kinetic characterization shows that pCPB behaves like other known basic carboxypeptidases. pCPB is more specific for substrates with C-terminal arginine than those with C-terminal lysine for all the natural and synthetic peptides tested. It also hydrolyzes the synthetic ester substrate more efficiently than the synthetic peptide substrate, especially at high pH. The active site Zn2+ can be replaced with other metals with change in substrate specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1995

Genomic structure, chromosomal localization, and conserved alternative splice forms of thrombopoietin.

Thrombopoietin (TPO), the ligand for c-mpl, is a novel cytokine comprising an amino terminal domain with homology to erythropoietin and a glycosylated carboxyl terminal domain that does not bear overall homology to other known proteins. We report the cloning of cDNAs encoding the porcine and murine TPO and the characterization of the human TPO gene. The cDNA for an additional splice form (TPO-2) with a four-amino-acid deletion within the erythropoietin-like domain has been isolated and is conserved between humans, pigs, and mice. Species comparison of TPO shows that the amino terminal erythropoietin-like domain is highly conserved, while the carboxyl terminal domain is less conserved. Recombinant murine TPO and human TPO are each able to activate both the murine and human c-mpl receptors, indicating an absence of strict species specificity. Human TPO is encoded by a single gene consisting of six exons and located on chromosome 3q27-28.

Alternative Splicing↗

PubMed1995

In vitro biotransformation of aflatoxin B1 (AFB1) in channel catfish liver.

The biotransformation of the dietary carcinogen aflatoxin B1 (AFB1) was examined in hepatic microsomal and cytosolic fractions from channel catfish, an aquatic species shown to be refractory to AFB1 toxicity and reported to be resistant to AFB1 hepatocarcinogenesis. Catfish liver microsomes catalyzed the in vitro oxidation of AFB1 to the reactive AFB1-8,9-epoxide (AFBO) at high substrate concentrations (128 microM AFB1) but not at low substrate concentrations (16 microM AFB1) which were more representative of environmental exposure. A similar trend was observed for the production of the hydroxylated metabolite aflatoxin M1 (AFM1). In contrast, hepatic microsomes prepared from rainbow trout, a species sensitive to AFB1 toxicity and hepatocarcinogenesis, activated AFB1 at 16 and 128 microM AFB1, with the rate of AFB1 epoxidation by trout microsomes exceeding that of catfish by more than sixfold. Treatment of channel catfish with 5,6-benzoflavone (beta NF, 20 mg/kg) resulted in a threefold increase in AFM1 formation but did not affect AFBO formation. AFB1 was rapidly reduced to aflatoxicol (AFL), a putative detoxification product of AFB1, at both low and high substrate concentrations. The rate of AFL production by channel catfish hepatic cytosol was 40- and 65-fold greater than observed for rainbow trout at 16 and 128 microM AFB1, respectively. Western blotting of catfish cytosols revealed the presence of a catfish cytosolic protein of approximately 25 kDa that displayed immunological cross-reactivity to rat GST Ya, but not to other rat or mouse alpha class GSTs with high AFBO-conjugating activity. Furthermore, catfish cytosolic GSTs did not catalyze the conjugation of AFBO with GSH. The results of these studies indicate that AFB1 is poorly oxidized by channel catfish microsomes, and suggest that the lack of microsomal AFB1 activation together with the rapid conversion of AFB1 to AFL contributes to the apparent resistance of channel catfish to AFB1 toxicity and hepatocarcinogenesis.

Aflatoxin B1↗

PubMed1995

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.

Animals↗

PubMed1995

Induction of phase I and phase II drug-metabolizing enzyme mRNA, protein, and activity by BHA, ethoxyquin, and oltipraz.

Various natural and synthetic compounds are known to protect against cancer by elevating phase II detoxification enzymes. Generally classified as monofunctional, these inducers are believed to trigger cellular signal(s) that activate gene transcription through an antioxidant or electrophile response element (ARE/EpRE) in responsive genes. In contrast, the phase I enzymes of drug metabolism (cytochrome P450s) are not believed to be induced by monofunctional inducers and P450 genes have not been found to contain functional ARE/EpREs. In this study, rats were treated with the monofunctional inducers tert-butylated hydroxyanisole, ethoxyquin, and oltipraz to study the inducibility of individual glutathione S-transferase isozymes, NADP(H):quinone oxidoreductase, gamma-glutamylcysteine synthetase, UDP-glucuronosyl transferase, and cytochrome P450 enzymes. Hepatic mRNAs were analyzed on Northern blots using gene-specific oligonucleotide probes for GST Ya1, Ya2, Yc1, Yc2, Yb1, Yb2, and Yf, for UGT 1*06, and for P450 1A1, 1A2, 2B1, 2C11, 3A2, and 4A1. NADP(H):quinone oxidoreductase and gamma-glutamylcysteine synthetase mRNAs were detected using cDNA probes. All the phase II detoxification enzymes analyzed, except GST Yf, were induced by the three monofunctional inducers, suggesting that these genes may be regulated by a mechanism involving an ARE/EpRE element in their promoter region. Interestingly, it was found that ethoxyquin was a particularly good inducer for both members of the P450 2B family, 2B1 and 2B2, and both ethoxyquin and oltipraz were also capable of modestly inducing P450 1A2 and 3A2. Oltipraz was found to slightly induce P450 2B2, but not 2B1, at the dose and time analyzed. Induction of mRNA generally correlated well with induction of protein levels determined by Western blot and/or enzyme activity measurements for selected enzymes. The results of this study suggest that many phase II enzymes may contain ARE/EpRE elements in addition to those confirmed to be regulated by a mechanism involving ARE/EpRE elements. In addition, it was found that several P450 enzymes were induced by monofunctional inducers, suggesting a possibility that some phase I enzymes may also be regulated by a mechanism involving ARE/EpRE elements.

Animals↗

PubMed1995

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.

Adolescent↗

PubMed1995

Role of cytochrome P4501A2 in chemical carcinogenesis: implications for human variability in expression and enzyme activity.

Cytochrome P4501A2 (CYP1A2) has been identified as a key factor in the metabolic activation of numerous chemical carcinogens, including aflatoxin B1, various heterocyclic and aromatic amines, and certain nitroaromatic compounds. In addition, CYP1A2 contributes to the inactivation of several common drugs and dietary constituents, including acetaminophen and caffeine. Two xenobiotic-responsive-element (XRE)-like sequences and an antioxidant response element (ARE) have been identified in the regulatory region of the CYP1A2 gene; however, the functionality of the ARE remains to be demonstrated. Based on in vivo phenotyping assays, substantial interindividual variability in CYP1A2 activity has been reported. Some population-based studies have reported either bi- or tri-modal distributions in CYP1A2 phenotype, suggesting a genetic basis for the large interindividual differences in CYP1A2 activity. However, despite the polymodal distributions reported for CYP1A2 activity, a distinct functional genetic polymorphism in the gene has not been identified. Potential mechanisms contributing to the large interindividual variability in CYP1A2 activity are discussed. A thorough understanding of the functions and regulation of the CYP1A2 gene may ultimately lead to new methods for preventing or intervening in the development of certain chemically-related human cancers.

Aflatoxin B1↗

PubMed1995

Thrombopoietin and the humoral regulation of thrombocytopoiesis.

In 1994 four biotechnology research groups reported the isolation and cloning of the ligand for the cytokine receptor c-Mpl and showed it to be the long-sought regulator of platelet production, thrombopoietin. Thrombopoietin is a hematopoietic growth factor of 332 amino acids composed of an amino terminal domain homologous to erythropoietin and a highly glycosylated carboxyl domain. The erythropoietin-like domain is the functional domain, whereas the glycosylated domain appears to stabilize circulating thrombopoietin. Thrombopoietin stimulates both proliferation of progenitor megakaryocytes and their maturation to platelet-producing megakaryocytes. Thrombopoietin induces dramatic increases in megakaryocyte number and platelet production in mice, indicating that it regulates both thrombopoiesis and megakaryocytopoiesis. Thrombopoietin also accelerates the recovery of platelets in myelosuppressed animals, suggesting that it will be clinically useful for the treatment of thrombocytopenia.

Animals↗

PubMed1995

Plasma carboxypeptidases as regulators of the plasminogen system.

Carboxy-terminal lysine residues on the surface of cells and fibrin bind plasminogen and control its activation. Since plasma contains basic carboxypeptidases, which remove carboxy-terminal lysines from protein substrates, we investigated if these enzymes are involved in the regulation of plasminogen binding sites. Plasma reduced plasminogen binding to cells, and this effect could be ascribed to the activity of the plasma carboxypeptidases. Purified carboxypeptidase N, which is constitutively active, and plasma carboxypeptidase B, which circulates as a zymogen, were both capable of significantly reducing plasminogen binding to cells. Dose titration experiments verified that plasma concentrations of either carboxypeptidase were sufficient to maximally affect plasminogen binding to cells. Furthermore, plasma carboxypeptidase B, but not carboxypeptidase N, reduced the rate of whole blood clot lysis induced by tissue-type plasminogen activator. These findings establish that plasma carboxypeptidases can modulate plasminogen binding to cells and control the rate of fibrinolysis. These functions delineate a novel role for the plasma carboxypeptidases in the regulation of the plasminogen system.

Blood Coagulation↗

PubMed1995

Zonal differences in DNA synthesis activity and cytochrome P450 gene expression in livers of male F344 rats treated with five nongenotoxic carcinogens.

Both increased cell proliferation and "altered" CYP gene expression are prominent phenomena associated with liver tumor promotion by nongenotoxic carcinogen treatment. To further characterize these two responses, groups of rats were kept on powdered rat chow diet containing 0.05% phenobarbital (PB) or 0.025% ciprofibrate (Cip) for 8 days or given 8 daily doses by gavage of pregnenolone 16 alpha-carbonitrile (PCN, 150 mg/kg/ml corn oil), 3,3',4,4'-tetrachlorobiphenyl (PCB-MC, 3 mg/kg/ml corn oil) or 2,2',4,4'-tetrachlorobiphenyl (PCB-PB, 7.5 mg/kg/ml corn oil). A minipump was implanted in the rat abdominal cavity to release bromodeoxyuridine (BRDU) 5 days after the start of nongenotoxic carcinogen treatment and the experiment was terminated 3 days later. BRDU-labeled parenchymal nuclei were observed primarily in the periportal area independent of nongenotoxic carcinogen treatment. Treatment with each of the 5 nongenotoxic carcinogens resulted in profound alterations in CYP gene expression at both the transcriptional and translational levels. Expression of CYP1A1, 1A1/2, 3A1, 2B1/2, and 4A immunoproteins demonstrated nongenotoxic carcinogen-specific patterns in both magnitude and zonal distribution. In agreement with the CYP immunoprotein data, treatment with each of the five nongenotoxic carcinogens resulted in a unique composition of mRNAs of CYP2B1, 2B2, 2C6, 2C11, 3A1, 3A2, and 4A1, which were variably increased or decreased relative to the untreated control livers, depending on the treatment. Similarly, the rate and pattern of CYP enzyme-mediated hydroxylation toward testosterone, 17 beta-estradiol, corticosterone, and lauric acid were greatly altered by nongenotoxic carcinogen treatment. According to the zonal distribution patterns of CYP immunoproteins, each hepatocyte in the cell plate from the periportal triad to the central vein has a characteristic and nongenotoxic carcinogen-specific composition of CYP enzymes. Because many endogenous substrates are modulators of DNA and RNA synthesis, intracellular kinetics of endogenous substrates of CYP enzymes in the corresponding hepatocytes could contribute, at least in part, to the differences in gene expression, differentiation, and cell proliferation among the hepatocytes in the cell plate.

Animals↗

PubMed1994

Enzymatic characteristics of chimeric mYc/rYc1 glutathione S-transferases.

Mice are resistant to aflatoxin carcinogenicity primarily due to expression of a glutathione S-transferase (mYc) with high catalytic activity toward aflatoxin B1-8,9-epoxide (AFBO). In contrast, rats are more sensitive to aflatoxin carcinogenicity due to the constitutive expression of a glutathione S-transferase with relatively low catalytic activity toward AFBO (rYc1). To identify the contribution of different regions of the mYc protein that confer high catalytic activity toward AFBO, six chimeric mYc/rYc1 GST enzymes were generated utilizing full and partial restriction enzyme digestions at two conserved StyI sites in the mYc and rYc1 complementary DNAs (between amino acid residues 56-57 and 142-143). Recombinant wild-type and chimeric glutathione S-transferases were bacterially expressed, affinity purified, and their catalytic activities measured toward AFBO, delta 5-androstene-3,17-dione, 1-chloro-2,4-dinitrobenzene, and ethacrynic acid. The set of chimeras displayed a wide range of catalytic activities toward the substrates assayed. The chimeras with the greatest activity toward AFBO were 1:56rat-57: 221mouse and 1:56mouse-57:142rat-143:221mouse, with AFBO conjugating activities 200 and 8 times greater than wild-type rYc1, respectively. These results demonstrate that the residues that confer high AFBO conjugation activity in mYc are located in the region spanning residues 57-221.

Aflatoxin B1↗

PubMed1994

Inhibition of cell proliferation by ciprofibrate in glutathione S-transferase P1-1-positive rat hepatic hyperplastic nodules.

Previous studies have demonstrated that short-term treatment with a peroxisome proliferator (PP) decreased the size and number of genotoxic carcinogen-induced hepatic hyperplastic lesions identified by gamma-glutamyl transpeptidase (GGT) or glutathione S-transferase P1-1 (rGSTP1-1) staining. However, longer-term PP treatment of animals bearing similar hepatic hyperplastic lesions produced an increase in both the size and number of liver tumors. To characterize the hepatic hyperplastic lesions which are inhibited or promoted by PP, a unique double labeling technique was developed to determine the relative rate of cell division (e.g., DNA synthesis) in rGSTP1-1-positive nodules before and after ciprofibrate (Cip) treatment. rGSTP1-1-positive nodules were induced with the Solt-Farber resistance protocol (diethylnitrosamine-2-acetylaminofluorene partial hepatectomy). Eleven weeks after diethylnitrosamine initiation, 3 groups of rats were maintained on a control chow diet or switched to a powdered chow diet containing 0.025% Cip or 0.05% phenobarbital (PB) for the last 8 days of the experiment. A minipump implanted in the abdominal cavity released [methyl-3H]thymidine continuously for 72 h and was then removed prior to CIp or PB treatment. A second minipump was then implanted which released bromodeoxyuridine to the abdominal cavity 5 days after the start of Cip or PB administration and lasted for 72 h until the termination of the experiment. Both the [methyl-3H]thymidine and bromodeoxyuridine labeling indices (LIs) were determined in the same group of cells within individual rGSTP1-1-positive nodules in the right posterior lobes of livers. PB treatment increased both the average number of persistent GGT-positive nodules and the ratio of persistent GGT-positive to rGSTP1-1-positive nodules/cm2. In contrast, Cip treatment greatly decreased the average number and area of persistent GGT-positive nodules, as well as the ratio between persistent GGT-positive and rGSTP1-1-positive nodules/cm2. Cip treatment also resulted in a 40% decrease in the average LI in the rGSTP1-1-positive nodules. In some rGSTP1-1-positive nodules, the LI was decreased from > 40% prior to Cip treatment to < 5% afterward, suggesting that Cip treatment interrupted progression in these nodules. Such drastic changes in the LI before and after treatments were not observed in either PB- or vehicle-treated (control) animals. A number of small nodules with a high bromodeoxyuridine LI but with no or very few [methyl-3H]thymidine-labeled nuclei and negative GGT and rGSTP1-1 staining were detected only in the Cip group.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

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)

Adolescent↗

PubMed1994

Enhancement of glutathione content in glutathione synthetase-deficient fibroblasts from a patient with 5-oxoprolinuria via metabolic cooperation with normal fibroblasts.

Fibroblasts from patients with the disease 5-oxoprolinuria have reduced glutathione synthetase activity and are thus glutathione (GSH) deficient. In this study, 5-oxoprolinuria fibroblasts (GM3877 cells) contained less GSH than normal diploid fibroblasts as determined by biochemical analysis and by flow cytometry using monochlorobimane. They also contained lower gamma-glutamylcysteine synthetase activity than normal cells. However, cocultures of GM3877 cells and normal cells displayed either normal or slightly elevated GSH content, depending upon the assay used. When differentially labeled with fluorescent beads, cocultured, and then isolated by fluorescence-activated cell sorting, both GM3877 cells and normal cells had GSH content similar to that of sorted normal cells cultured alone, whereas sorted GM3877 cells cultured alone showed depressed GSH content. GM3877 cells had detectable levels of gamma-glutamylcysteine (gamma-GC) when cultured alone, but gamma-GC was undetectable in these cells when they were cocultured with normal cells, indicating that it was efficiently metabolized to GSH by the normal cells. These changes in low-molecular-weight thiols were likely to have been mediated by metabolic cooperation across gap junctions because they were dependent upon confluency and because media conditioned by either cell type failed to significantly alter the GSH content of the other cell type. Cocultures exposed to moderate levels of hydrogen peroxide showed less depletion of GSH than GM3877 cells cultured alone, suggesting that the sharing of low-molecular-weight thiols or other reductants via metabolic cooperation can protect cells from oxidative stress.

Amino Acid Metabolism, Inborn Errors↗

PubMed1994

Increase in gamma-glutamylcysteine synthetase activity as a mechanism for butylated hydroxyanisole-mediated elevation of hepatic glutathione.

Previous studies have demonstrated that dietary administration of butylated hydroxyanisole (BHA) and other phenolic antioxidants increases hepatic glutathione (GSH). The purpose of this study was to examine whether BHA increases GSH by increasing the activity of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in hepatic GSH biosynthesis. Male Swiss-Webster mice were fed BHA in the diet at various doses (0.05-0.75%, w/w, of diet) for 14 days. An additional study examined the effects of 0.75% BHA on hepatic GSH and GCS activity at 1, 4, 8, and 14 days, and at various times following cessation of the BHA diet. BHA increased both GSH and GCS activity in a dose- and time-dependent fashion. At the maximal dose of 0.75% BHA, hepatic GSH and GCS activity was increased by 1.5-fold and 2.1-fold, respectively, by Day 8, and remained at this level at Day 14. GSH was initially depleted at 1 day on the BHA diet, but had returned to control levels at Day 4. Upon removal of the BHA diet, both GCS and GSH returned to control values within 4 days. Hepatic cytosolic GCS activity from BHA-treated mice was inhibited by GSH in a manner similar to that of GCS from untreated mice. These data demonstrate that GCS activity is increased by BHA, and that this increase may be responsible for the elevation of hepatic GSH after BHA treatment observed previously. Whether the BHA-mediated increase in GCS activity is the result of enhanced transcriptional activation of the GCS gene, or results from stabilization of existing GCS enzyme activity, requires further investigation.

Animals↗

PubMed1994

Modulation of gamma-glutamylcysteine synthetase large subunit mRNA expression by butylated hydroxyanisole.

Dietary 2(3)-tert-butyl-4-hydroxyanisole (BHA) treatment has been shown to increase hepatic glutathione (GSH) content in rats and mice. Subsequent studies in our laboratory have demonstrated that hepatic gamma-glutamylcysteine synthetase (GCS) activity is increased in mice treated with dietary BHA. To test whether this increase in GCS activity follows an increase in hepatic messenger RNA for the large subunit of GCS (GCS-LS mRNA), a 390-base pair fragment corresponding to a region near the 5' end of the rat GCS-LS cDNA sequence was amplified using the PCR reaction and used to detect GCS-LS mRNA on Northern blots. Hepatic GSH, GCS activity, and GCS-LS mRNA levels were determined either in mice treated with BHA in the diet for 12 days or mice injected with diethyl maleate (DEM), phorone, and/or DL-buthionine-[S,R]-sulfoximine (BSO) over a 24 hr period. BHA caused a 1.5-fold increase in GSH levels, a 1.7-fold increase in hepatic GCS activity by Day 12, and a rapid 5-fold increase in hepatic GCS mRNA levels reaching maximal levels after 2-3 days. Partial depletion of GSH with either phorone (70%) or DEM (50%) resulted in a 4- to 5-fold increase in hepatic GCS-LS mRNA levels by 9 hr and a 1.5- to 2-fold increase in hepatic GSH and GCS activity by 24 hr. Depletion of GSH with the GCS enzyme inhibitor BSO had no effect on GCS mRNA expression, even though GSH was depleted to 30%. When BSO was combined with the phorone treatment GSH levels were depleted to < 10%, but the large increase in GCS-LS mRNA seen with phorone alone was greatly attenuated. These data suggest that depletion of GSH per se, is not sufficient to induce elevation of GCS-LS mRNA levels, but that the formation of GSH conjugates may be required to trigger GCS-LS mRNA induction. The increase in GCS-LS mRNA levels may account for the increase in GCS activity and elevation of GSH observed following BHA treatment, as well as the "rebound" of GSH above control levels observed 18-24 hr following depletion of GSH by other chemicals. These results are consistent with the Michael acceptor, hypothesis by Talalay.

Animals↗