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PubMed2004

Modern diagnostics in chronic myeloproliferative diseases (CMPDs).

According to the new WHO classification a group of chronic myeloproliferative diseases (CMPDs) were defined: chronic myeloid leukemia (CML), chronic neutrophilic leukemia (CNL), chronic eosinophilic leukemia and hypereosinophilic syndrome (CEL/HES), polycythemia vera (PV), chronic idiopathic myelofibrosis (with extramedullary hematopoiesis, CIMF), essential thrombocythemia (ET), and so called CMPD/unclassifiable. As clinical features and laboratory findings differ widely between these diseases several diagnostic approaches are mandatory at diagnosis for classification and are needed also for follow up studies, especially for the measurement of minimal residual disease (MRD). We here outline the laboratory set up at diagnosis and during follow up in CMPDs with specific focus on the respective therapeutical consequences. Only by using a comprehensive diagnostic panel including cytomorphology, cytogenetics, and molecular genetic methods establishing the correct diagnosis, optimizing treatment as well as evaluating treatment response is possible in CMPDs today.

Blood Cell Count↗
PubMed1999

Induction of drug metabolizing enzymes by 1,7-phenanthroline and oltipraz in mice is unrelated to Ah-responsiveness.

The protective effect of several classes of compounds against the toxic and neoplastic effects of xenobiotics has been attributed to the induction of noncytochrome P450 (P450) drug metabolizing enzymes. Glutathione S-transferases (GST), NAD(P)H: quinone oxidoreductase (QOR), and UDP-glucuronosyltransferases (UGT) play a prominent role in detoxification and can be induced by oltipraz and other N-heterocyclic compounds in rats. In contrast to the induction of these enzymes by aryl hydrocarbon (Ah)-receptor agonists, induction by oltipraz and 1,7-phenanthroline is not accompanied by CYP1A induction. This study investigated the induction of drug metabolizing enzymes following administration of oltipraz and 1,7-phenanthroline in four mouse strains (C57B6A-J, Frings x C57B6J, Frings, CF-1) exhibiting varying degrees of responsiveness to an Ah-receptor agonist. The relative Ah responsiveness was determined in all strains by the induction of hepatic Cypla after three daily doses of 3-methylcholanthrene (20 mg/kg). After treatment with 1,7-phenanthroline and oltipraz (150 mg/kg i.g.) daily for 3 days, all strains showed similar induction of GST and QOR activities for each inducer. Both compounds were equally effective in elevating GST activity, but 1,7-phenanthroline was more effective than oltipraz in elevating QOR activity. In addition to GST and QOR changes, 1,7-phenanthroline significantly elevated UGT (1-naphthol) activity in the Frings strain. Neither compound produced significant changes in Cypla parameters. The independence of 1,7-phenanthroline and oltipraz induction of GST and QOR from Cypla-responsiveness is in line with the concept that N-heterocycle-containing inducers act by mechanisms other than an Ah-receptor-dependent pathway in which the P450 response has been masked or prevented.

Animals↗
PubMed2002

Oral versus injectable ovulation induction agents for unexplained subfertility.

BACKGROUND: Oral (anti-oestrogens) and injectable (gonadotrophins) ovulation induction agents have been used to increase the number of eggs produced by a woman per cycle in treatment for unexplained subfertility. It is unclear whether there are significant advantages of one type of treatment over the other in this context or in terms of fertility. OBJECTIVES: To assess the efficacy of oral versus injectable ovulation induction agents for unexplained subfertility. SEARCH STRATEGY: The search strategy of the Menstrual Disorders and Subfertility Group was used for the identification of relevant randomised controlled trials. SELECTION CRITERIA: All trials where oral ovulation induction agents were compared with injectable ovulation induction agents in treatment groups generated by randomisation, from couples with unexplained subfertility, were considered for inclusion in the review. DATA COLLECTION AND ANALYSIS: Five randomised controlled trials, including a total of 231 identified couples with unexplained subfertility, were found and included in this review. All trials were assessed for quality criteria. The studied outcomes were pregnancy, live birth, miscarriage, multiple birth, occurrence of ovarian hyperstimulation syndrome and cycle cancellation. MAIN RESULTS: Where trials with important co-interventions were excluded, there was no significant difference in the odds of beneficial outcomes for oral versus injectable ovulation induction agents - live birth per couple (OR 0.06, 95%CI 0.00-1.15), pregnancy per woman (OR 0.33, 95%CI 0.09-1.20); nor of detrimental outcomes for injectable versus oral agents - miscarriage (OR 0.11, 95%CI 0.00-2.84); there were no reported cases of multiple births, cases of ovarian hyperstimulation or discontinued cycles consequent upon overstimulation. Where trials with the co-intervention of a human chorionic gonadotrophin trigger injection (given only in the injectable ovulation induction agent treatment arm) were not excluded there was no significant difference in the odds of live birth per couple (OR 0.40, 95%CI 0.15-1.08). However oral ovulation induction agents had significantly reduced odds of pregnancy per woman compared to injectable ovulation induction agents (OR 0.41, 95%CI 0.17-0.80). For detrimental outcomes, there were no significant differences in the odds of miscarriage (OR 0.61, 95%CI 0.09-4.01) and multiple birth (OR 1.08, 95%CI 0.16-7.03) for injectable versus oral agents. No data were available concerning the occurrence of ovarian hyperstimulation syndrome nor cycle cancellation. REVIEWER'S CONCLUSIONS: There is insufficient evidence to suggest that oral agents are inferior or superior to injectable agents in the treatment of unexplained subfertility. Information on harms is sketchy, and remains compatible with large differences in either direction. Much larger trials than have previously been undertaken are required to provide information on relative harms as well as benefits.

Administration, Oral↗
PubMed2000

Interleukin-1 induction of collagenase 3 (matrix metalloproteinase 13) gene expression in chondrocytes requires p38, c-Jun N-terminal kinase, and nuclear factor kappaB: differential regulation of collagenase 1 and collagenase 3.

OBJECTIVE: To examine the mechanism of interleukin-1 (IL-1)-induced collagenase 3 (matrix metalloproteinase 13 [MMP-13]) gene expression in cultured chondrocytes for the purpose of better understanding how the gene is induced in these cells, and how it contributes to cartilage degradation in osteoarthritis. METHODS: The transcriptional and posttranscriptional responses of the MMP-13 gene to IL-1 were assessed first. Then, direct inhibitors of mitogen-activated protein kinase (MAPK) signaling pathways and a constitutive repressor of nuclear factor kappaB (NF-kappaB) were used to assess the role of each pathway in IL-1-mediated induction of MMP-13. RESULTS: We found that IL-1 induction of MMP-13 requires p38 activity, c-Jun N-terminal kinase (JNK) activity and NF-kappaB translocation. These results suggest that both NF-kappaB and activator protein 1 transcription factors are necessary for IL-1 induction of MMP-13. We also compared the signaling pathways necessary for IL-1 to stimulate collagenase 1 (MMP-1) in articular chondrocytes and chondrosarcoma cells and found that IL-1 induction of MMP-1 requires different pathways from those required by MMP-13. In chondrosarcoma cells, MMP-1 induction depends on p38 and MEK (an MAPK kinase of the extracellular signal-regulated kinase pathway) and does not require JNK or NF-kappaB. In articular chondrocytes, inhibition of MEK had no effect, while inhibition of p38 gave variable results. CONCLUSION: These studies demonstrate, for the first time, that p38, JNK, and NF-kappaB are required for IL-1 induction of MMP-13. The results also highlight the differential requirements for signaling pathways in the induction of MMP-1 and MMP-13. Additionally, they demonstrate that induction of MMP-1 by IL-1 in chondrocytic cells depends on unique combinations of signaling pathways that are cell type-specific.

Chondrocytes↗
PubMed2001

Early carbonic anhydrase induction in the gills of the blue crab, Callinectes sapidus, during low salinity acclimation is independent of ornithine decarboxylase activity.

Carbonic anhydrase (CA) induction in the gills of the euryhaline blue crab, Callinectes sapidus, was measured in response to lowered environmental salinity. Simultaneous measurements of ornithine decarboxylase (ODC) activity were made in gills and nonbranchial tissues to determine whether ODC activity and the resultant synthesis of polyamines played a role in the initiation and regulation of CA induction. CA induction in the seventh gill pair (G7) was proportional to the decrease in ambient salinity, but activity in the third gill pair (G3) remained unchanged. Induction began by 24 hr after low salinity transfer, much earlier than previously reported, and peaked after 4 days. The magnitude of salinity change affected the magnitude of CA induction only, not the time course. A general cell volume regulatory response, as measured by the appearance of total ninhydrin-positive substances (TNPS) in the hemolymph, was initiated within 4 hr of low salinity transfer and was complete by 24 hr post-transfer. General cell swelling may be the initial signal in the pathway of CA induction. ODC activity in the gills of acclimated animals was not influenced by salinity. For crabs transferred from 35 to 25 ppt, ODC activity did not change significantly over the time course of acclimation. There was an early but transient increase in ODC activity in all tissues for crabs acclimated to 28 ppt and transferred to 15 ppt. Induction of ODC activity does not appear to be a precursor for CA induction; therefore, it does not appear that polyamines are substantially involved in the up-regulation of transport enzyme activity in low salinity. ODC, and resultant polyamine synthesis, may, however, have a role in cell volume regulation.

Adaptation, Physiological↗
PubMed1993

Influence of extracellular matrix overlay on phenobarbital-mediated induction of CYP2B1, 2B2, and 3A1 genes in primary adult rat hepatocyte culture.

To obtain efficient induction by phenobarbital (PB) of cytochrome P450 (CYP) genes in primary hepatocyte culture, previous studies have demonstrated the importance of culturing hepatocytes on a substratum derived from extracellular matrix (ECM or Matrigel), or in highly enriched medium formulations such as Chee's. In the present study, we have reexamined a variety of hepatocyte culture conditions and their relative abilities in preserving the PB-induction response within the CYP2B and 3A gene subfamilies. A modified culture system was developed that combines a highly effective medium formulation in conjunction with dilute concentrations of a Matrigel overlay. Specifically, hepatocytes were attached to a Collagen I substratum and overlaid with either daily (50 micrograms/ml medium) or single (233 micrograms/ml) additions of Matrigel. The PB-induction response obtained in vitro closely resembled that occurring in vivo. Induction in culture by 1 mM PB of CYP2B1, 2B2, and 3A1 mRNA levels was highly dependent on a variety of factors, including medium formulation and 0.1 microM dexamethasone addition. The response to dexamethasone addition on this gene battery varied in a medium-specific manner. Cells maintained a higher level of PB-induction response when maintained in Williams' E medium than with Chee's, Waymouth's, or Ultraculture media. The kinetics of PB induction also were more rapid in cells cultured in Williams' E medium. PB exposures in Chee's medium resulted in elevation of two CYP2B-immunoreactive proteins as detected by Western blot analysis, together with increased rates of O-dealkylation of benzyloxy- and pentoxyresorufin. However, Chee's formulation produced an abnormal PB-induced expression of CYP1A1, as determined by mRNA analysis, high rates of O-dealkylation of 7-ethoxyresorufin, and inhibition of enzymatic activity by 1 microM alpha-naphthoflavone. This paradoxical expression of CYP1A1 was not observed in PB-treated cultures grown in Williams' E medium. Thus, these studies demonstrated that the use of a physiologically balanced medium, i.e., Williams' E formulation, together with an overlay of ECM, preserves PB-induction responsiveness closely resembling that observed in vivo, and should better facilitate mechanistic investigations into the molecular nature of PB induction.

Animals↗
PubMed1997

Characterization of a pretranscriptional pathway for induction by phenobarbital of cytochrome P450 3A23 in primary cultures of adult rat hepatocytes.

Our laboratory has proposed that phenobarbital (PB), a typical lipophilic agent that induces some members of the supergene family of liver microsomal cytochromes P450 (e.g., CYP2B1/2 and CYP3A23), acts through a complex process inhibitable by the presence of growth hormone (GH), the absence of some components of the extracellular matrix, or a disrupted cytoskeleton. To verify that these manipulations of the culture environment block specific steps in the PB induction pathway rather than simply exerting nonspecific or toxic effects on CYP2B1/2 gene transcription, we have now examined PB induction of CYP3A23, a gene known to also be transcriptionally activated by dexamethasone (DEX) through a "nonclassical" pathway apparently involving the glucocorticoid receptor. We found that in primary cultures of adult rat hepatocytes treated with PB, induction of CYP3A23 mRNA, just as we reported for induction of CYP2B1/2 mRNA, required the use of Matrigel (a reconstituted basement membrane) and was blocked by the presence of cytoskeletal inhibitors (colchicine or cytochalasins) or of physiologic concentrations of GH in the culture medium. Moreover, PB induction of CYP3A23 and of CYP2B1/2 mRNAs was greatly diminished by inhibitors of cAMP-dependent protein kinase (PKA). In striking contrast, induction of CYP3A23 mRNA by DEX was unaffected by any of these alterations of the culture conditions that block its induction by PB. We conclude that the effects of extracellular matrix, GH, disruption of the cytoskeleton, and activation of cAMP-dependent protein kinase, pharmacologically define multiple, pretranscriptional steps in the pathway(s) for PB induction of liver cytochromes P450.

Animals↗
PubMed1996

Role of cytokines, tyrosine kinase, and protein kinase C on production of superoxide and induction of scavenging enzymes in human leukocytes.

We investigated the effects of proximal modulators of cytokines, tyrosine kinase (TK), and protein kinase C (PKC) on reactive oxygen species (ROS) generation and the induction of scavenging enzymes, superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH-Px) of human neutrophils and lymphocytes, by using IL1-alpha, TNF-alpha, and IFN-gamma and neutralizing antibodies to these cytokines. Inhibitors of TK (ST638 and herbimycin) or PKC (H-7, calphostin, and staurosporine) were also used. The results revealed that both (O2)- generation stimulated by five different agents (opsonized zymosan, A23187, PAF, PMA, and fMLP) and the inductions of all three scavenging enzymes were potentiated by priming with TNF-alpha. In contrast, both (O2)- generation and enzyme induction were attenuated by priming with IL1-alpha, with the exception of PMA-stimulated (O2)- generation. IFN-gamma decreased (O2)- generation but increased scavenging enzyme induction. Antibodies to all three cytokines and all the TK and PKC inhibitors decreased (O2)- stimulated by most agents, but markedly enhanced (O2)- levels stimulated by PAF. Induction of all three enzymes was enhanced equally by low concentrations of each of the three anticytokine antibodies, while each of the TK or PKC inhibitors decreased induction of SOD and GSH-Px and increased catalase induction. These results suggest that both ROS generation and scavenging enzyme induction are controlled in complex ways by the actions of these three proximal mediators. This supports our hypothesis that disturbances in the regulation of early events of cell activation can lead to oxidative tissue injury.

Adult↗
PubMed1999

Protein synthesis-dependent and -independent induction of p69 2'-5'-oligoadenylate synthetase by interferon-alpha.

The 2'-5'-oligoadenylate (2-5A) synthetases are a family of interferon-alpha (IFN-alpha) inducible enzymes that block viral replication by activating a latent endonuclease during viral infections. In Ramos cells, induction of mRNAs for the intermediate isoform of 2-5A synthetase (p69) requires five-fold higher IFN-alpha than is required for induction of the small isoform (p40). The p40 and p69 isoforms are similarly induced between 1 and 24 h with maximal induction at 8 h. At 48 h, however, p69 is more strongly induced than p40. Induction of p69 and p40 between 1 and 24 h is protein synthesis independent whereas at 48 h, p69 induction becomes dependent on protein synthesis. Initial induction of both isoforms requires tyrosine kinase activation and is enhanced by activation of a separate signalling pathway by the tumour promoter, 12-0-tetradecanoyl phorbol-13-acetate (TPA). These data suggest that induction of the p40 is predominantly protein synthesis independent, whereas p69 induction occurs in two phases, an initial protein synthesis independent phase and a delayed protein synthesis dependent phase.

2',5'-Oligoadenylate Synthetase↗
PubMed1994

Regulation of cytochrome P4501A1 in teleosts: sustained induction of CYP1A1 mRNA, protein, and catalytic activity by 2,3,7,8-tetrachlorodibenzofuran in the marine fish Stenotomus chrysops.

Cytochrome P4501A1 (CYP1A1) is known to play important roles in the activation and detoxification of carcinogens and other toxicants in vertebrate animals, including fish. Although extensively studied in mammalian systems, the regulation of CYP1A forms in other vertebrates is less well understood. We examined the time course and dose-response relationships for induction of CYP1A1 mRNA, protein, and catalytic activity by 2,3,7,8-tetrachlorodibenzofuran (TCDF) in the marine fish Stenotomus chrysops (scup). The time course of CYP1A1 induction was determined following a single ip dose (10 nmol/kg) of 2,3,7,8-TCDF. Hepatic ethoxyresorufin O-deethylase activity was increased after 1 day, reached a maximum by 8 days, and was still elevated 14 days after treatment. The content of immunodetectable CYP1A1 protein in liver was elevated on Day 1 and continued to increase through 14 days. CYP1A1 protein content was also strongly induced in heart and gill beginning at 2 days after treatment and extending through Day 14. Hepatic CYP1A1 mRNA was strongly induced by 1 day after dosing and remained elevated through 14 days. The sustained induction of CYP1A1 mRNA by 2,3,7,8-TCDF contrasts with the transient induction seen previously in fish treated with nonhalogenated inducers and most likely reflects differences in persistence of the inducers. Dose-response studies indicated that induction of CYP1A1 mRNA, protein, and catalytic activity occurred following doses of 2,3,7,8-TCDF as low as 0.4 nmol/kg (120 ng/kg), within the range of whole-body contents of this congener measured in fish from contaminated environments. The estimated dose producing half-maximal CYP1A1 induction in scup was approximately 2-10 nmol/kg, suggesting that the sensitivity of these fish to induction may be as great as or greater than that of rats. In contrast to previous results obtained with 3,3',4,4'-tetrachlorobiphenyl (TCB) and beta-naphthoflavone, which appear to inhibit or inactivate CYP1A1 in fish and other vertebrates, there was a good correlation among levels of CYP1A1 mRNA, protein, and catalytic activity in individual fish following various doses of 2,3,7,8-TCDF. The difference in response to 2,3,7,8-TCDF versus 3,3',4,4'-TCB may reflect differences in the inducing potencies of the two compounds relative to their similar potencies as inhibitors of CYP1A1 catalytic activity. In additional studies to evaluate structure-activity relationships for CYP1A1 induction by chlorinated dibenzofurans in fish, scup were treated with 2,3,6,8-tetrachlorodibenzofuran (2,3,6,8-TCDF). At 10 or 50 nmol/kg, 2,3,6,8-TCDF was inactive as an inducer of CYP1A1 mRNA, protein, or catalytic activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Relative potencies of induction of hepatic drug-metabolizing enzyme genes by individual PCB congeners.

The induction of a variety of drug-metabolizing enzymes by polychlorinated biphenyl (PCB) congeners that elicit a 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD)-type hepatic pleiotropic response, including 2,3,3',4,4'-pentachlorobiphenyl (BZ 105), 2,3',4,4',5-pentachlorobiphenyl (BZ 118), 2,3,3',4,4',5-hexachlorobiphenyl (BZ 156), and 3,3',4,4',5,5'-hexachlorobiphenyl (BZ 169) was examined. Following dietary exposure to the individual congeners for 5 days, livers were removed and catalytic assays for cytochrome P450 (CYP) isozymes 1A1 and 1A2 were performed. Additionally, total cellular RNA coding for hepatic drug-metabolizing genes (CYP 1A1, CYP 1A2, microsomal epoxide hydrolase, glutathione S-transferase [GST] Ya/Yc, and the TCDD-inducible isozyme of aldehyde dehydrogenase [ALDH] was quantified. 3-Methylcholanthrene (MC), TCDD, or BZ 156 (32 ppm) caused nearly maximal induction of the CYP 1A proteins but lower induction of the other genes. When the dose-response curves for induction of various drug-metabolizing genes (CYP 1A1 and 1A2, microsomal epoxide hydrolase, the GST Ya/Yc subfamily and ALDH) were examined, a spectrum of ED50s (half-maximal inductions) was observed. While CYP 1A2 exhibited an ED50 of 1.7 ppm, the induction of ALDH was shifted far to the right (ED50 > 11 ppm). Thus, different genes in a single tissue may display different dose-response characteristics. The potency (extent of induction of CYP 1A1 activity resulting from a given dietary dose) was BZ 169 >> BZ 156 > BZ 118 > BZ 105. In contrast, the potencies of the four congeners for CYP 1A1 induction were nearly equivalent when related to hepatic PCB burden, apparently due to the preferential accumulation in the liver of BZs 169 and 156 following low-level administration in the diet.

Animals↗
PubMed1999

Time- and concentration-dependent induction of CYP1A1 and CYP1A2 in precision-cut rat liver slices incubated in dynamic organ culture in the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin.

In a previous 24-h study, precision-cut rat liver slices were validated as a useful in vitro model for assessing the dose-related induction of CYP1A1 and CYP1A2 in rat liver following exposure to 2, 3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Further assessment of the utility of this model was accomplished by initially exposing rat liver slices to medium containing TCDD (0.01 nM) for 24 h and incubating the slices up to an additional 72 h in TCDD-free medium. The slices remained viable throughout the incubation period with an intracellular potassium content varying from 45.2 +/- 2.3 micromol/g at 48 h to 50.0 +/- 1.6 micromol/g at 72 h. In TCDD-exposed slices, CYP1A1 protein and its respective enzymatic activity, the O-deethylation of ethoxyresorufin (EROD), significantly increased with time over the 96-h incubation period, with EROD activity increasing from 63.6 +/- 14.2 at 24 h to 905 +/- 291 pmol/mg/min at 96 h. Under identical incubation conditions, but in the absence of TCDD, the EROD activity for the control liver slices ranged from 14. 3 +/- 4.3 to 44.9 +/- 11.9 pmol/min/mg. Conversely, the level of CYP1A2 protein and its respective activity (acetanilide hydroxylation) transiently decreased from 24 to 96 h with no significant differences observed between the control (0 nM TCDD) and treatment group (0.01 nM TCDD). The concentration-effect relationship at 96 h was characterized by incubating rat liver slices for the initial 24 h in medium containing TCDD at concentrations ranging from 0.1 pM to 10 nM. Induction of CYP1A1 protein and EROD activity was observed for all treatment groups with the 10 nM TCDD treatment group displaying greater than 100-fold induction compared to control (0 nM TCDD). Immunohistochemical localization of CYP1A1 protein within liver slices supported the time- and concentration-dependent induction of EROD activity by TCDD. The induction of CYP1A1 was initially observed to be centrilobular, with increased expression due to both elevated CYP1A1 within cells and the recruitment of additional cells expressing CYP1A1 throughout the entire liver slice. Additionally, the immunohistochemical analysis of the liver slices demonstrated the conservation of tissue architecture following up to 96 h of incubation in dynamic organ culture and provided further evidence for maintenance of tissue viability. In comparison to CYP1A1, the induction of CYP1A2 at 96 h was a less sensitive response, with significant induction of CYP1A2 protein and its respective activity occurring at a medium concentration of 0.1 nM TCDD (686 pg/g liver). In general, increasing the incubation period from 24 to 96 h markedly increased TCDD-induced expression of CYP1A1 and minimally enhanced CYP1A2 expression. Moreover, extending the incubation period to 96 h resulted in in vitro induction profiles for CYP1A1 and CYP1A2 that were qualitatively and quantitatively similar to that previously observed following in vivo exposure to TCDD (Drahushuk et al., Toxicol. Appl. Pharmacol. 140, 393-403, 1996).

Animals↗
PubMed2001

Effect of metals on polycyclic aromatic hydrocarbon induction of CYP1A1 and CYP1A2 in human hepatocyte cultures.

Environmental cocontamination by polycyclic aromatic hydrocarbons (PAHs) and metals could affect the carcinogenic consequences of PAH exposure by modifying PAH induction of PAH-bioactivating CYP1A. The effect of As, Pb, Hg, or Cd (ranked as the most hazardous environmental metals by EPA and ATSDR) on CYP1A1 and 1A2 induction by benzo[a]pyrene (BaP), benzo[b]fluoranthene (BbF), dibenzo[a,h]anthracene (DBahA), benzo[a]anthracene (BaA), and benzo[k]fluoranthene (BkF) has thus been investigated in fresh human hepatocyte cultures. Induction was probed by ethoxyresorufin-O-deethylase activity, by immunoblots, and by RT-PCR. Uptake of PAHs into the hepatocytes varied according to PAH and liver donor: 84% of 5 microM BaA and 25-40% of 5 microM DBahA was taken up in 24 h. Hepatocytes retained viability up to 1 microM Cd and 5 microM Pb, Hg, or As and 5 microM PAHs. PAH induction of CYP1A in hepatocytes was variable, some cultures expressed CYP1A1 and others CYP1A1 and 1A2, and to variable extents. Induction efficiency (relative to DMSO controls) at 2.5 microM PAH concentration was in the order BkF (7.6-fold) > DBahA (6.1 fold) > BaP (5.7 fold) > BbF (3.9-fold) > BaA (2.5-fold). All four metals (1-5 microM) decreased CYP1A1/1A2 induction by some of the PAHs with dose-, metal-, and PAH-dependency. Arsenic (5 microM) decreased induction by 47% for BaP, 68% for BaA, 45% for BbF, 79% for BkF, and 53% for DBahA. Induced CYP1A2 protein was much more extensively decreased than 1A1 protein, and CYP1A2 mRNA and, to variable extents, CYP1A1 mRNA were decreased by As. Thus the metals in PAH/metal mixtures could diminish PAH carcinogenicity by decreasing induction of their bioactivation by CYP1A1/1A2.

Arsenic↗
PubMed1987

The effects of aging on phosphofructokinase induction during lymphocyte mitogenesis in relation to DNA and protein synthesis.

The incorporation of radiolabeled leucine into phytohemagglutinin-stimulated human lymphocytes increases by 9 hours after mitogen addition in the young whereas this process is delayed by two-fold in the aged (18 hours). Once induced, the leucine incorporation is about 56% less in the aged as compared to the young. The induction of phosphofructokinase (PFK) catalytic activity mimics the induction of protein synthesis in both young (9 hours) and aged (18 hours) subjects also taking twice as long to induce in the aged and attaining much lower levels of induction with increasing subject age. The increase of thymidine incorporation in mitogen-stimulated cells does not occur until 12 hours after the increase in leucine incorporation in both the young (21 hours) and aged (30 hours) which also represents a 9 hour age-related delay in induction. The marked increase in protein synthesis rate occurs in a concerted manner with the induction of glycolysis and the delay and impairment in protein biosynthesis in the aged appears to relate to the similar age-related findings for glycolytic enzyme induction. The mitogen-induced increase in DNA synthesis is a later event and the age-related delay in DNA synthesis induction may be secondary to the delay in the induction of protein synthesis. Other enzyme-dependent processes besides DNA synthesis and glycolysis may also be secondary to a primary slowing of protein synthesis in the aged and related to the delayed cell cycle time frequently observed in aged subjects.

Adult↗
PubMed1984

Studies on the mechanisms of induction of N-nitrosodimethylamine demethylase by fasting, acetone, and ethanol.

Previous work has shown that induction of a high-affinity NADPH-dependent nitrosodimethylamine demethylase (NDMAd) in liver microsomes occurs in rats due to fasting, ethanol consumption, and streptozotocin-induced diabetes. Several lines of observations suggest that this is due to the induction of specific cytochrome P-450 isozymes. Induction of P-450 species by ethanol has also been observed by other investigators. Since each of the above altered metabolic states has in common elevated levels of ketone bodies, the possible role of acetone, a known inducer of NDMAd, in the induction of the demethylase activity was investigated. Levels of endogenous acetone in fasted rats correlated (r = 0.72) with a three- to fourfold increase in NDMAd activity. However, a dose-response experiment showed endogenous levels of acetone to be capable of causing at most 40% of the induction in fasted rats. This suggests that other ketone bodies or factors may have contributed to the induction. The induction of NDMAd by ethanol was enhanced by alcohol dehydrogenase inhibitors pyrazole and acetaldehyde oxime, suggesting that ethanol, rather than its metabolites, was responsible for the induction.

Acetone↗
PubMed1991

Heme oxygenase induction by CoCl2, Co-protoporphyrin IX, phenylhydrazine, and diamide: evidence for oxidative stress involvement.

The induction of heme oxygenase in rat liver by cobaltous chloride (CoCl2) and Co-protoporphyrin IX is entirely prevented by the administration of alpha-tocopherol and allopurinol. CoCl2 was converted in the liver into Co-protoporphyrin IX before it induced heme oxygenase activity. Actinomycin and cycloheximide affected to a similar degree the induction of heme oxygenase by both CoCl2 and Co-protoporphyrin IX. Administration of either CoCl2 or Co-protoporphyrin strongly decreased the intrahepatic GSH pool, a decrease which was completely prevented by the administration of either alpha-tocopherol or allopurinol. The latter compounds prevented heme oxygenase induction as well as the decrease in hepatic GSH when administered 2 h before, together with, or 2 h after CoCl2. However, when given 5 h after administration of CoCl2, alpha-tocopherol and allopurinol showed no preventive effect. Similar results were obtained when Co-protoporphyrin IX was used, with the difference that when alpha-tocopherol and allopurinol were given 2 h after administration of the inducer, they showed no protective effect. Phenylhydrazine and diamide also induced heme oxygenase activity in rat liver. This inductive effect was preceded by a decrease in the intrahepatic GSH pool, which took place several hours before induction of the oxygenase. Administration of alpha-tocopherol and allopurinol prevented induction of the oxygenase but had no effect on the decrease in GSH levels. These results suggest that the induction of heme oxygenase by phenylhydrazine and the diamide is preceded by an oxidative stress which very likely originates in the depletion of GSH. The induction of heme oxygenase by hemin was not prevented by administration of alpha-tocopherol or allopurinol. Coprotoporphyrin IX did not affect the pattern of the molecular forms of hepatic biliverdin reductase, at variance with CoCl2, which is known to convert molecular form 1 of the enzyme into molecular form 3.

Allopurinol↗
PubMed1994

Pretranslational induction of cytochrome P4501A enzymes by beta-naphthoflavone and 3-methylcholanthrene occurs in different liver zones.

Most of the cytochrome P450 (CYP) genes are expressed in an uneven, zonated pattern in the liver. Factors regulating this regionally restricted expression are not well known. In this study we have analysed cell lysates obtained from opposite zones of rat liver by infusing digitonin to the perfused liver to study the zonation of CYP1A1 and CYP1A2 induction. 3-Methylcholanthrene induced CYP1A1 protein in perivenous cells, while a low dose of beta-naphthoflavone caused periportal induction. Analysis of CYP1A1 mRNA from cell lysates by reverse transcriptase-coupled polymerase chain reaction (RT-PCR) and in situ hybridization experiments both demonstrated that this inducer-specific differently localized effect occurred at the pretranslational level. A corresponding difference in the regional pattern of CYP1A2 induction was seen: induction by beta-naphthoflavone reversed the constitutive perivenous pattern into a periportal CYP1A2 mRNA pattern while induction after 3-methylcholanthrene treatment was more panacinar. Attempts to identify the regiospecific factors involved were made by comparing the in vitro induction of CYP1A1 by beta-naphthoflavone and 3-methylcholanthrene in hepatocytes isolated from the periportal and perivenous region. However, after isolation, induction seemed to be independent of the source of the cells. Our results demonstrate the existence in the liver of regionally acting factors that mediate the induction of CYP1A1 and 1A2 in a local and inducer-specific fashion. These factors could be Ah receptor associated binding proteins operating in vivo, but no longer in isolated cells.

Animals↗
PubMed1994

Characterization of cytochrome P450 2E1 induction in a rat hepatoma FGC-4 cell model by ethanol.

The hepatic microsomal ethanol-oxidizing system (MEOS) has been well characterized as an important pathway in ethanol metabolism. Cytochrome P450 2E1 (CYP 2E1), the principal component of MEOS, is ethanol inducible and has been implicated in hepatotoxicity associated with alcohol abuse and exposure to organic solvents. Results of chronic in vivo experiments have shown that ethanol induction of hepatic CYP 2E1 occurs by a two-step mechanism. The first step of induction is associated with low blood alcohol concentrations (BACs) and appears to be post-transcriptional, whereas high BACs observed in step-two induction are associated with increased CYP 2E1 gene transcription. The mechanisms underlying these induction steps are under intense investigation. Progress in this area has been limited due to lack of hepatic cell culture models that express CYP 2E1. We report here an in vitro tissue culture cell model, the FGC-4 hepatoma cell line, that exhibits basal levels of CYP 2E1 apoprotein that are inducible by ethanol treatment. Total cellular RNA and microsomal fractions were isolated from control or ethanol-treated confluent cells, and CYP 2E1 mRNA and apoprotein levels were characterized by northern blot or immunoblot analysis, respectively. Initial experiments on isolated microsomes revealed detectable levels of CYP 2E1 apoprotein in control cells that were induced 5-fold in cells treated with 100 mM ethanol for 24 hr. Concentration-response experiments demonstrated that the maximal 24-hr induction in CYP 2E1 apoprotein level was 5-fold and was attained at a concentration of 10 mM ethanol. Interestingly, while the steady-state mRNA levels encoding CYP 2E1 were detectable, they remained unchanged in identically treated cells. Furthermore, there was no observed increase in CYP 2E1 mRNA levels in an extended time course to 72 hr or at higher alcohol concentrations (up to 1500 mM), providing preliminary evidence that the induction is post-transcriptional. The time course of CYP 2E1 apoprotein induction by exposure to 100 mM ethanol demonstrated maximal induction at 8 hr. Measurement of CYP 2E1 apoprotein levels after removal of ethanol from pretreated cells demonstrated the half-life of the apoprotein to be 12.7 hr, in good agreement with previous reports using primary hepatocytes. The half-life of the induced protein after ethanol removal in the presence of cyclohexamide (10 micrograms/mL) was biphasic with a rapid 1.8 hr first phase followed by a slower 44.7 hr second phase.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗