Cytochrome P4501A Levels Research Articles

Differential response to benzo[a]pyrene in human lung adenocarcinoma cell lines: The absence of aryl hydrocarbon receptor activation

Benzo[a]pyrene (B[a]P) has been shown to produce DNA adducts and to initiate pulmonary carcinogenesis in animals. We observed differential susceptibility to B[a]P in two human lung adenocarcinoma cell lines, A427 and CL3. DNA adducts were induced by B[a]P treatment in CL3 cells, however, A427 cells were much less responsive to B[a]P treatment. Cytochrome P450 1A1 (CYP1A1) is involved in bioactivation of B[a]P in nonhepatic tissues. Cotreatment with α-naphthoflavone, a CYP1A1 inhibitor, abolished DNA adduct formation by B[a]P in CL3 cells. Nevertheless, CYP1A1 inducer β-naphthoflavone, enhanced DNA adduct formation by B[a]P in both A427 and CL3 cells. Both enzyme activity and mRNA levels of CYP1A1 were highly induced by 1 or 10 μM B[a]P treatment for 24 hr in CL3 cells but not in A427 cells. Protein levels of AhR and aryl hydrocarbon receptor nuclear translocator (Arnt) were similar in A427 and CL3 cells before B[a]P treatment. However, B[a]P induced a retarded band with the [ 32P]-dioxin responsive element in CL3 cells, but not in A427 cells. This study demonstrated that variation in AhR-mediated CYP1A1 induction contributes to differential susceptibility to B[a]P-DNA adduct formation in human lung cells. Since AhR and/or Arnt function is impaired in A427 cells, this cell line offers a model for investigating the repression mechanisms of CYP1A1 induction by B[a]P in lung cells.

Modulation of Cytochrome P-450 and Glutathione S-Transferase Isoform Expression In Vivo by Intact and Degraded Indolyl Glucosinolates

Various dietary substances modulate the xenobiotic metabolism and may thereby protect against toxicity and carcinogenicity of food toxins. The effects of pure indolyl glucosinolates, which are present in cruciferous vegetables, on induction of specific cytochrome P-450 (CYP) and glutathione S-transferase (GST) isoforms have not been studied previously. In the present study, glucobrassicin (GB) and neoglucobrassicin (NeoGB) were purified from broccoli by use of a single-column method. Furthermore, a mixture containing 48% GB, 36% NeoGB, and 16% 4-methoxyglucobrassicin was obtained. The modulatory effects of the pure GB, NeoGB, and the mixture on activities and levels of hepatic CYP 1A, 2B1/2, and 2E1 and α- and μ-GST isoforms were investigated in male Wistar rats. The indolyl mixture was the most powerful and NeoGB the weakest inducer of microsomal hepatic CYP 1A1 protein and 7-ethoxyresorufin O-deethylase activity. Furthermore, intact indolyl glucosinolates were more powerful inducers than the in vitro myrosinase-degraded indolyl glucosinolates. The hepatic 7-methoxyresorufin O-deethylase activities, but not CYP 1A2 protein, were induced by pure GB, whereas the mixture and NeoGB showed only minor effects. Neither CYP 2B1/2 nor 2E1 was induced by the indolyl glucosinolates. None of the hepatic GST subunits analyzed, rGST A1/2, A3, or M3, was induced significantly by the purified indolyl glucosinolates.

Elevated levels of multiple cytochrome P450 forms in tilapia from Billings Reservoir-São Paulo, Brazil

Cytochrome P4501A (CYP1A) levels in tissues of fish inhabiting polluted areas have been used extensively in biomonitoring studies in Europe and North America. However, little information is available about the extent of CYP1A expression in fish from South American waters, nor on the expression of other CYP proteins in fish from polluted sites. The content of total cytochrome P450 and b 5 and of proteins cross-reactive with antibodies to teleost (scup) CYP1A-, CYP2B- and CYP3A-like proteins, as well as rates of alkoxy-resorufin- O-dealkylases (ARODs) with methoxy-, ethoxy-, pentoxy- and benzyloxyresorufin substrates, were analyzed in liver and kidney microsomes from tilapia ( Oreochromis niloticus) from a reference site (Estação de Piscicultura de Pindamonhangaba, São Paulo) and a polluted site (Billings Reservoir, São Paulo, Brazil). Levels of total microsomal P450 and b 5 were elevated 1.8- and 3.3-fold, respectively, in liver and 4.3- and 2.4-fold in kidney of tilapia from Billings Reservoir. Rates of all four ARODs and the levels of CYP1A-, CYP2B- and CYP3A-cross-reactive proteins were elevated in liver and kidney of fish from Billings Reservoir compared to those in reference fish. Hepatic microsomal EROD and MROD were about 20-fold greater and levels of 1A protein more than 400-fold greater in Billings fish. Antibodies to teleost CYP1A and putative CYP3A recognized single bands while Pab to the scup CYP2B-like protein recognized three protein bands in liver and kidney. This suggests that there are multiple CYP2B-like proteins expressed in tilapia. Immunohistochemical analysis also indicated increased expression of CYP1A cross-reactive proteins in cells of liver, gill, kidney and heart. The apparent induction of CYP1A probably reflects the exposure to aryl hydrocarbon receptor agonists present as pollutants in the Billings Reservoir. Elevated levels of the other CYP forms in tissues of tilapia from Billings Reservoir could be related either to the presence of pollutants or to some natural compound present in the diet, since tilapia is known as an herbivorous species.

Elevated levels of cytochrome P4501A (CYP1A) in ringed seals from the Baltic Sea

The high frequency of a variety of disorders observed in Baltic seals have been correlated with elevated concentrations of organochlorines. For this study, we determined hepatic CYP1A activities and apoprotein levels in ringed seals ( Phoca hispida) exposed to high levels of organochlorides in the Baltic Sea and in corresponding seals from a relatively unpolluted area (Svalbard Region). Activities of ethoxyresorufin deethylase (EROD) and pentoxyresorufin depentylase (PROD) in seal liver microsomes were increased about 3-fold in the Baltic sample, the EROD activity reaching similar levels to those found in 2,3,7,8-tetrachlorodibenzo- p-dioxin-induced rats (about 1,2 nmol/mg protein per min). No sex-related differences in EROD or PROD activities were observed in either population. Hepatic CYP1A levels in ringed seals were further characterised by inhibition and Western blot analysis. α-Naphthoflavone and furafylline were used as chemical inhibitors, having highly specific inhibiting effects on CYP1A1 and CYP1A2, respectively, in rat and human liver. α-Naphthoflavone inhibited potently both EROD and PROD activities, while inhibition by furafylline was weaker. A monoclonal antibody specific to CYP1A in the rat (MAb 1-7-1), used in the immunoblots, revealed a substantially increased amount of CYP1A protein in the Baltic seals. CYP2B-related proteins were not detected in any seal sample using MAb 2-66-3, which is specific for rat CYP2B. Thus, both inhibition and Western blotting experiments indicate that CYP1A is mainly responsible for the marked increase in microsomal EROD and PROD activities in the livers of Baltic ringed seals. Since the properties of all CYP enzymes vary between species, further studies are needed to verify whether CYP1A1 or CYP1A2 mediates the observed EROD induction in the ringed seal.

Variation in levels of cytochrome P4501A, 2B, 2E, 3A and 4A-immunopositive proteins in digestive gland of indigenous and transplanted mussel Mytilus galloprovincialis in Venice Lagoon, Italy

Mytilus galloprovincialis digestive gland microsomes were prepared from (i) indigenous populations sampled from a clean reference (Lido) and an urban-contaminated site (Salute) and (ii) mussels transplanted for up to 3 weeks from Lido to an industrial-contaminated site (CVE) in Venice Lagoon, Italy. Western blot analysis was performed using antibodies to five mammalian or fish CYP forms (1A, 2B, 2E, 3A, 4A). Simultaneously run M. edulis digestive gland partially purified CYP aided identification of immunopositive bands. Levels of CYP1A, CYP2E, and CYP4A-immunopositive proteins were 50 to 300% higher in indigenous M. galloprovincialis from Salute compared to Lido ( p < 0.05). Three weeks after transplantation to CVE, levels of only the CYP1A-immunopositive protein were determined to be higher (63%) than levels for Lido ( p < 0.05), indicating that anti-CYP1A shows greater specificity for a contaminant-inducible CYP form than the other antibodies.

Role of nitroreductases but not cytochromes P450 in the metabolic activation of 1-nitropyrene in the HepG2 human hepatoblastoma cell line

1-Nitropyrene is an environmental contaminant that is mutagenic in many prokaryotic and eukaryotic systems, including the hypoxanthine-guanosine phosphoribosyl transferase (HGPRT) locus in the human hepatoma cell line HepG2. Metabolism and DNA adduct formation of [ 3H]1-nitropyrene in the HepG2 were quantified to understand the role of nitroreduction and/or cytochrome P450-mediated C-oxidation of 1-nitropyrene in DNA adduct formation and mutagenicity. In uninduced HepG2 cells, 10 μM [ 3H]1-nitropyrene was metabolized principally by nitroreduction to 1-aminopyrene (516 pmol/24 hr/10 6 cells), and by cytochrome P450-mediated C-oxidation to K-region trans-dihydrodiols (37 pmol/24 hr/10 6 cells), 1-nitropyren-3-ol (51 pmol/24 hr/10 6 cells), and l-nitropyren-6-ol and 1-nitropyren-8-ol (77 pmol/24 hr/10 6 cells). Pretreatment of the HepG2 cells for 24 hr with 5 nM 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) resulted in a complete change in the metabolism of [ 3H]1-nitropyrene, with 1-nitropyren-6-ol and l-nitropyren-8-ol formation (449 pmol/24 hr/10 6 cells) being 80-fold greater than 1-aminopyrene formation (6 pmol/24 hr/10 6 cells). This increase in C-oxidation of 1-nitropyrene was consistent with increased levels of cytochrome P450 1A. The only DNA adduct detected using the 32P-postlabeling assay in the HepG2 cells administered 1-nitropyrene was N-(2′-deoxyguanosin-8-yl)-1-aminopyrene (dG-C8-AP). Induction of C-oxidative metabolism through TCDD treatment resulted in a concomitant decrease in dG-C8-AP formation. DNA adducts for oxidized 1-nitropyrene metabolites were not detected in the TCDD-treated HepG2 cells administered 1-nitropyrene, which indicates that cytochrome P450-mediated C-oxidative pathways are detoxification pathways in HepG2 cells.

Exposure of male american plaice (Hippoglossoides platessoides) to contaminated marine sediments decreases the hatching success of their progeny

AbstractThere is increasing concern over the effect environmental contaminants may be having on male vertebrate reproduction, particularly persistent organics. This study sought to determine the effects of marine sediments, contaminated with organic compounds, on the reproductive performance of male American plaice (Hippoglossoides platessoides). Maturing fish were exposed to contaminated sediments, in the laboratory, for a 5‐month period leading up to spawning. Sediments were obtained from three known environmentally contaminated sites, in which site 1 was the most toxic, followed by site 2, with site 3 relatively uncontaminated (i.e., control). Semen collected from all males with motile sperm were used to fertilize eggs from a single nonsediment‐exposed female. Eggs fertilized with sperm from males exposed to sediments from site 1 produced 48% fewer larvae (p &lt; 0.05), compared to the control group (i.e., site 3). Similarly, males exposed to site 2 produced 35% fewer larvae, relative to the control. There were no differences between groups with respect to sperm number, seminal sodium and potassium concentrations, and gonadosomatic index. The levels of liver cytochrome P450 1A1 (CYP1A1) mRNA, used as a biomarker for exposure to organics, were threefold higher in fish exposed to site 1 sediments, and twofold higher in fish exposed to site 2 sediments, relative to the site 3‐exposed controls, at the end of the experiment. A significant negative correlation (r = −0.65, n = 16, p &lt; 0.007) exists between male CYP1A1 levels and hatch success, with increasing CYP1A1 levels corresponding to decreased numbers of hatched larvae. These data indicate that long‐term exposure of male American plaice to marine sediments contaminated with organics can result in a reduction in the number of eggs hatched, which correlates with increased levels of hepatic CYP1A1.

Multienzyme-mediated stable and transient multidrug resistance and collateral sensitivity induced by xenobiotics.

Determinants of cellular sensitivity to anticancer drugs include enzymes that catalyze their biotransformation. Coordinated induction of some of these enzymes is known to be caused by a number of dietary constituents, environmental contaminants, pharmacological agents and other xenobiotics, e.g. 3-methylcholanthrene and catechol. Despite the potential for inducing simultaneous changes in tumor cell sensitivity to a wide range of drugs, scant attention has been paid to the impact that dietary constituents and other xenobiotics might have on the therapeutic outcome of cancer chemotherapy. The aim of this investigation was to demonstrate the potential of xenobiotic-induced multienzyme-mediated stable and transient multidrug resistance/collateral sensitivity in a model system. Human breast adenocarcinoma MCF-7/0 cells and a stably oxazaphosphorine-resistant subline thereof, MCF-7/OAP, were grown in the presence of 3-methylcholanthrene (3 microM), catechol (30 microM), or vehicle for 5 days. Spectrophotometric and spectrofluorometric assays were used to quantify catalytic activities and thus cellular levels of cytosolic class 3 aldehyde dehydrogenase, glutathione S-transferase, DT-diaphorase, UDP-glucuronosyl transferase and cytochrome P450 1A1. A colony-forming assay was used to quantify cellular sensitivities to several anticancer drugs. Relative to their untreated counterparts, MCF-7/0 and MCF-7/OAP cells treated with 3-methylcholanthrene or catechol transiently expressed elevated levels of cytosolic class 3 aldehyde dehydrogenase, glutathione S-transferase, DT-diaphorase and UDP-glucuronosyl transferase, and were transiently, more resistant to mafosfamide, melphalan, and mitoxantrone, and more sensitive to EO9. Further, MCF-7/0 and MCF-7/OAP cells treated with 3-methylcholanthrene, but not those treated with catechol, transiently expressed elevated levels of cytochrome P450 1A1 and were transiently more sensitive to ellipticine. Relative to MCF-7/0 cells, MCF-7/OAP cells stably overexpressed all but cytochrome P450 1A1 and were stably, more resistant to mafosfamide, melphalan and mitoxantrone, and more sensitive to EO9. Inclusion of relatively specific inhibitors of, or alternative substrates for, the enzymes of interest during drug exposure negated the influence of enzyme overexpression on cellular sensitivities to these agents. Untreated, and 3-methylcholanthrene- or catechol-treated, MCF-7/0 and MCF-7/OAP cells were equisensitive to vincristine and nearly so to doxorubicin. Collectively, these experiments illustrate the potential for both stable and transient xenobiotic-induced multienzyme-mediated multidrug resistance/collateral sensitivity that, although also the result of a single event, is mechanistically different from, and pertains to a largely different group of anticancer agents than does, the multidrug resistance caused by cell surface multidrug transporters.

Induction of Cytochrome P4501A in the Intertidal Fish Anoplarchus purpurescens by Prudhoe Bay Crude Oil and Environmental Induction in Fish from Prince William Sound

Cytochrome P4501A (CYP1A) induction is a sensitive and specific adaptive response in fish exposed to xenobiotics including petroleum hydrocarbons. CYP1A expression was examined in the intertidal fish Anoplarchus purpurescens collected from or caged at reference sites and sites oiled by the Exxon Valdez spill in Prince William Sound. Immunoblotting of hepatic microsomes showed that the content of CYP1A in fish at oiled sites was up to 6-fold greater than that in reference site fish. Fish injected with the CYP1A inducer β-naphthoflavone showed a 70-fold induction of CYP1A, to levels six times the highest level seen in the field. To model the field exposure, fish were maintained over oiled sediments and/or fed amphipods collected from an oiled site. Hepatic microsomal CYP1A was induced 49-fold in fish exposed to oiled sediments but rapidly returned to control levels after fish were removed from oil exposure. Immunohistochemistry showed CYP1A induction in multiple organs. CYP1A staining in hepatic and some extrahepatic cells was highly correlated (r 2 ≥ 0.95) with the hepatic CYP1A content detected by immunoblot. Oiled food induced CYP1A most strongly in intestinal mucosal epithelial and endothelial cells. Relatively low levels of CYP1A were observed in liver, gill, and gonad of fish exposed to oil through the diet, consistent with the metabolism of dietary hydrocarbons by intestinal CYP1A. Exposure to oiled sediment alone strongly induced CYP1A in endothelial cells in all organs examined. Thus, oil present in Prince William Sound sediments more than 1 year after the spill was able to induce CYP1A in intertidal fish. The caging and laboratory experiments indicate that the induction of CYP1A observed in field specimens of A. purpurescens from oiled sites was due primarily to persistent spill-derived hydrocarbons.

A Comparison of Cytochrome P4501A (CYP1A) mRNA Inducibility in Four Species of Atlantic Coast Anadromous Fishes

Quantification of levels of cytochrome P4501A (CYP1A) gene expression in sentinel species of fishes has been proposed as a management tool to evaluate contamination of aquatic systems. Based on preliminary studies, we hypothesized that differences in CYP1A mRNA inducibility among individuals, populations, or species might lead to spurious conclusions when using this approach in environmental monitoring programs. To address this possibility, we quantitated and compared CYP1A mRNA induction levels in four species of common Atlantic Coast estuarine fish: smooth flounder, hogchoker, striped bass, and Atlantic tomcod, which were treated with model chemicals (beta naphthoflavone (β-NF), or benzo[a]pyrene at 10 ppm) known to induce CYP1A mRNA, or were exposed to contaminated environments. Species-specific CYP1A DNA probes were generated from PCR (polymerase chain reaction) amplification of genomic DNA using conserved oligonucleotide primers, and, along with cloned rainbow trout and Atlantic tomcod CYP1A cDNA probes were used to quantify CYP1A mRNA levels in northern blot analyses. Successful PCR amplification of CYP1A hybridizable DNA fragments was observed for all four species. Results from northern blot analyses showed large differences in CYP1A mRNA induction among species; only Atlantic tomcod exhibited significant induction of CYP1A mRNA for both chemically treated (97-fold) and environmentally exposed fish (34-fold). Significant, although lower, levels of induction were observed in β-NF treated (14-fold) smooth flounder, but not in environmentally exposed smooth flounder. Only low levels (not significant) of CYP1A gene induction were detected in hogchokers and striped bass. We conclude that CYP1A mRNA inducibility differed significantly among fish taxa perhaps due to differences in regulation of gene expression, suggesting that careful selection of sentinel species should be exercised prior to the use of CYP1A mRNA induction in environmental monitoring programs. However, the significance of differences in CYP1A mRNA inducibility in relation to higher level biological endpoints has yet to be determined.

Temporal induction pattern of hepatic cytochrome P450 1A in thermally acclimated dab ( Limanda limanda) treated with 3,3′,4,4′-Tetrachlorobiphenyl (CB77)

Mature male dab ( Limanda limanda) acclimated at 10° and 16°C were orally administered a single dose of 0.5 mg/kg 3,3′,4,4′-tetrachlorobiphenyl (CB77). At both temperatures, levels of cytochrome P450 1A (CYP1A) protein and 7-ethoxyresorufin O-deethylase (EROD) activity showed a two to six fold induction 40 days after CB77 treatment compared to control groups. Maximum responses of both EROD activity and CYP1A protein for the warm-acclimated fish were observed at 5 days after treatment. For the cold-acclimated fish a slow, progressive elevation for both EROD activity and CYP1A protein was observed and maximum responses were measured 40 days after treatment. Absolute EROD activity and CYPIA protein levels of fish from both temperatures were equally high at 40 days after treatment. Since in the control groups EROD activity and CYP1A protein levels were higher in the cold-acclimated fish, the magnitude of induction was higher in the warm acclimated ones. The highest concentrations of CB77 in muscle of fish from both temperatures were found at 5 and 10 days after treatment. The liver somatic index (LSI) showed 1.5 fold significantly higher values for the fish acclimated at 10°C.

Induction of CYP1A1 and ALDH-3 in Lymphoid Tissues from Fisher 344 Rats Exposed to 2,3,7,8-Tetrachlorodibenzodioxin (TCDD)

The immune system is a primary target for toxic insult by a number of drugs and environmental chemicals, many of which require activation to toxic metabolites by drug-metabolizing enzymes. We compared the induction of drug-metabolizing enzymes, including cytochrome P450 1A1 (CYP1A1) and aldehyde dehydrogenase (ALDH), in lymphoid tissues of F344 rats following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). ALDH was induced in both the spleen and the thymus after TCDD treatment, with maximal expression at 9 and 15 days, respectively. Thymic microsomal preparations from TCDD-treated animals expressed elevated levels of inducible CYP1A1 as compared to microsomes from the spleens of treated animals or tissues from control rats. TCDD treatment also resulted in increased ethoxyresorufin-O-deethylase (EROD) activity in the thymus. There were no detectable mRNA transcripts for CYP1A1 in peripheral blood or splenic lymphocytes from treated animals; however, CYP1A1 transcripts were induced in isolated thymocytes, whole spleen, and whole thymus.In vitroexposure to TCDD did not result in induction of immunoreactive CYP1A1 in thymocytes unless simultaneously activated with the mitogen, phytohemagluttinin (PHA). Immunohistochemical localization of CYP1A1 in immune tissues indicated that cells other than the lymphoid populations are responsible for the increased CYP1A1 expression. The pattern of CYP1A1 induction was related to the expression of the Ah receptor (AhR) in immune tissues. Western blot analyses demonstrated less AhR present in peripheral blood lymphoid cells and spleen, as compared to whole tissues. These studies indicate that while drug-metabolizing enzymes are present in immune tissues, the induction of enzymes is selective in different lymphoid cells.

Comparative assessment of metabolic enzyme levels in macrophage populations of the F344 rat

The immune system is a direct target for toxic insult by a number of drugs and other chemicals, many of which require activation to toxic metabolites by drug-metabolizing enzymes. We compared the induction of drug-metabolizing enzymes, including cytochrome P450 1A1 (CYP1A1) and aldehyde dehydrogenase (ALDH), which are differentially expressed in various macrophage populations following treatment of F344 rats with the inducer 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). Kupffer cells, alveolar macrophages and splenic macrophages from TCDD-treated animals expressed elevated levels of inducible CYP1A1 as compared to other macrophage subpopulations or cells from control rats. TCDD treatment also resulted in increased ethoxyresorufin- O-deethylase (EROD) activity and total cytochrome P450 content in tissue-derived macrophages. Immuno-reactive protein and mRNA transcripts for CYP1A1 were not detectable in resident peritoneal macrophages or peripheral blood monocytes. Examination of aromatic hydrocarbon receptor (AhR) levels in macrophage populations suggests that the ability of TCDD to induce metabolic enzymes in specific cell types correlates well with AhR expression. In vivo activation of macrophages, using either Bacillus of Calmette and Guérin, Mycobacterium tuberculosis (BCG) or polyinosinic:polycytidylic acid (Poly I:C), caused no significant alteration in the levels of induction of CYP1A1. ALDH-3 induction was similar in all macrophage populations examined. These studies indicate that macrophages, particularly those from portals of entry, may be induced to produce increased levels of specific enzymes, and the induction is dependent upon their maturational stage rather than their activation state. The metabolism of xenobiotics to toxic intermediates by immune cells and its role in immunosuppression are discussed.

Hepatic cytochrome P4501A induction in DAB (Limanda limanda) after oral dosing with the polychlorinated biphenyl mixture clophen A40

AbstractThe flatfish dab (Limanda limanda) serves as an indicator species in pollution monitoring programmes in the North Sea. The present study investigated the induction response of the monooxygenase system and haematological changes in female dab after multiple administrations of a technical mixture of polychlorinated biphenyls (PCBs). Mature female dab were dosed with 1 mg of the PCB mixture Clophen A40 (Clo A40) in sunflower oil every 6 weeks, with a maximum of three doses per fish. In all PCB‐administered groups, levels of cytochrome P4501A (CYP1A) protein, measured with a semi‐quantitative ELISA method, and 7‐ethoxyresorufin O‐deethylase (EROD) activity showed a three‐ to ninefold induction 14 d after dosing compared with control groups, smaller but also significant increases were observed in total cytochrome P450 (Σ P450) levels. Although the PCB concentrations and the corresponding toxic equivalent (TEQ) value in muscle tissue still increased after administration of the second and third dose of Clo A40, maximum responses of the EROD activity were already reached after the first dose at a TEQ value for chlorinated biphenyls (CB‐TEQ) of 2 ng/g lipid. The PCB patterns of liver and muscle tissue of female dab from the central North Sea were found to be virtually identical. Hence, the use of PCB concentrations in muscle as a qualitative model for changes in the liver appears legitimate. Haemoglobin concentrations were elevated after the third dose of Clo A40, whereas haematocrit values and the mean corpuscular haemoglobin concentration (MCHC) between treated and control groups did not differ.

Relationship between graft cytochrome P-450 3A content and early morbidity after liver transplantation.

Cytochrome P-450 3A metabolizes CsA into several metabolites with very limited pharmacological activity and toxicity. In 40 liver donors, the relative concentrations of P-450 3A was assessed by immunoblot analysis using a specific monoclonal antibody exhibited a 10-fold variation (mean = 92 +/- 50 arbitrary units [AU]/mg) in levels. Problems consequent upon CsA usage (nephrotoxicity, neurotoxicity, and hypertension) occurred in 8 of 34 transplant recipients in the immediate postoperative period, and in these 8 patients the problems were always associated with low graft P-4503A levels (mean = 52 +/- 19 A.U./mg, P = 0.0003, cf. patients with no toxicity). Four of these patients had CsA levels that were also high, but 4 had CsA levels in the therapeutic range. Episodes of early graft rejection were related to higher P-450 3A levels (mean = 110 +/- 24 A.U./mg). Cytochrome P-450 3A levels were also found to be inversely related to CsA whole blood levels (assessed with a specific antibody 12 hr after the intravenous infusion of CsA at 1 mg/kg in the recipients (P < 0.02). Cytochrome P-450 3A can provide information that should allow individualized immunosuppression with CsA maintaining therapeutic levels but avoiding toxicity in susceptible individuals.

CYP1A1 mRNA levels as a human exposure biomarker: use of quantitative polymerase chain reaction to measure CYP1A1 expression in human peripheral blood lymphocytes.

Accurate human risk assessment requires sensitive methods to evaluate dose-response relationships, especially following low level exposures. We have developed a reverse transcriptase polymerase chain reaction (RT-PCR) method to quantitative cytochrome P450-1A1 (CYP1A1) mRNA levels in human blood lymphocytes. Many polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene, and chlorinated PAH such as polychlorinated dibenzodioxins, dibenzofurans and biphenyls induce CYP1A1 expression through activation of an endogenous protein, the Ah receptor. Using a quantitative competitive RT-PCR method that included a synthetic internal standard we determined copy numbers of CYP1A1 mRNA in resting as well as mitogen-stimulated human blood lymphocytes. In mitogen-stimulated human blood lymphocytes assay variation was approximately 10% for measurement of this low expression gene and mRNA levels correlated well with ethoxyresorufin-O-deethylase (EROD) activity. The expression of mRNA was induced 20-fold upon culturing human lymphocytes with 10 nM TCDD. In nonstimulated, uninduced lymphocytes CYP1A1 levels are extremely low (1000 copies mRNA/10(4) cells) and cannot be measured by EROD activity. Studies of CYP1A1 mRNA expression in chemically-exposed populations are in progress.