P450 1A1 mRNA Levels Research Articles

Metabolic Detoxication Pathways for Sterigmatocystin in Primary Tracheal Epithelial Cells

Human health effects of inhaled mycotoxins remain poorly documented, despite the large amounts present in bioaerosols. Among these mycotoxins, sterigmatocystin is one of the most prevalent. Our aim was to study the metabolism and cellular consequences of sterigmatocystin once it is in contact with the airway epithelium. Metabolites were analyzed first in vitro, using recombinant P450 1A1, 1A2, 2A6, 2A13, and 3A4 enzymes, and subsequently in porcine tracheal epithelial cell (PTEC) primary cultures at an air-liquid interface. Expressed enzymes and PTECs were exposed to sterigmatocystin, uniformly enriched with (13)C to confirm the relationship between sterigmatocystin and metabolites. Induction of the expression of xenobiotic-metabolizing enzymes upon sterigmatocystin exposure was examined by real-time quantitative real-time polymerase chain reaction. Incubation of 50 μM sterigmatocystin with recombinant P450 1A1 led to the formation of three metabolites: monohydroxy-sterigmatocystin (M1), dihydroxy-sterigmatocystin (M2), and one glutathione adduct (M3), the latter after the formation of a transient epoxide. Recombinant P450 1A2 also led to M1 and M3. P450 3A4 led to only M3. In PTEC, 1 μM sterigmatocystin metabolism resulted in a glucuro conjugate (M4) mainly excreted at the basal side of cells. If PTEC were treated with β-naphthoflavone prior to sterigmatocystin incubation, two other products were detected, i.e., a sulfo conjugate (M5) and a glucoro conjugate (M6) of hydroxy-sterigmatocystin. Exposure of PTEC for 24 h to 1 μM sterigmatocystin induced an 18-fold increase in the mRNA levels of P450 1A1, without significantly induced 7-ethoxyresorufin O-deethylation activity. These data suggest that sterigmatocystin is mainly detoxified and is unable to produce significant amounts of reactive epoxide metabolites in respiratory cells. However, sterigmatocystin increases the P450 1A1 mRNA levels with unknown long-term consequences. These in vitro results obtained in the porcine pulmonary tract need to be confirmed in human epithelial cells.

Accelerated DNA adduct formation in the lung of the Nrf2 knockout mouse exposed to diesel exhaust.

Diesel exhaust (DE) has been recognized as a noxious mutagen and/or carcinogen, because its components can form DNA adducts. Mechanisms governing the susceptibility to DE and the efficiency of such DNA adduct formation require clarification. The transcription factor Nrf2 is essential for inducible and/or constitutive expression of a group of detoxification and antioxidant enzymes, and we hypothesized that the nrf2 gene knockout mouse might serve as an excellent model system for analyzing DE toxicity. To address this hypothesis, lungs from nrf2(-/-) and nrf2(+/-) mice were examined for the production of xenobiotic-DNA adducts after exposure to DE (3 mg/m(3) suspended particulate matter) for 4 weeks. Whereas the relative adduct levels (RAL) were significantly increased in the lungs of both nrf2(+/-) and nrf2(-/-) mice upon exposure to DE, the increase of RAL in the lungs from nrf2(-/-) mice exposed to DE were approximately 2.3-fold higher than that of nrf2(+/-) mice exposed to DE. In contrast, cytochrome P4501A1 mRNA levels in the nrf2(-/-) mouse lungs were similar to those in the nrf2(+/-) mouse lungs even after exposure to DE, suggesting that suppressed activity of phase II drug-metabolizing enzymes is important in giving rise to the increased level of DNA adducts in the Nrf2-null mutant mouse subjected to DE. Importantly, severe hyperplasia and accumulation of the oxidative DNA adduct 8-hydroxydeoxyguanosine were observed in the bronchial epidermis of nrf2(-/-) mice following DE exposure. These results demonstrate the increased susceptibility of the nrf2 germ line mutant mouse to DE exposure and indicate the nrf2 gene knockout mouse may represent a valuable model for the assessment of respiratory DE toxicity.

Cytokine-mediated suppression of cytochrome P450 1A1 in Hepa-1c1c7 cells by pokeweed mitogen

This study investigated the effects of pokeweed mitogen (PWM) on the regulation of cytochrome P450 (P450) 1A1 expression in an in vitro model, using murine hepatoma cell line Hepa-1c1c7 and murine macrophage cell line RAW 264.7 cell cultures. PWM added directly to Hepa-1c1c7 cells had no effect on 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)-induced P450 1A1-specific 7-ethoxyresorufin O-deethylase (EROD) activity. However, TCDD-induced EROD activity and P450 1A1 mRNA levels were markedly suppressed when Hepa-1c1c7 cells were cultured with PWM-treated conditioned media from RAW 264.7 in a dose-dependent manner. Concomitant treatment with PWM and pentoxifylline, a TNFα synthesis inhibitor, to RAW 264.7 cells decreased the suppressive effects of PWM on TCDD-induced EROD activity. In PWM-exposed RAW 264.7 cell cultures, TNFα and IL-6 levels increased in a dose-dependent fashion. When antibodies to TNFα or/and IL-6 were added to PWM-treated conditioned media from RAW 264.7, the suppression of EROD activity was inhibited. These results suggested the suppression of P450 1A1 by PWM was mediated exclusively by TNFα and IL-6, released from macrophages.

Toxicity of 2, 3, 7, 8‐tetrachlorodibenzo‐p‐dioxin to early life stage brook trout (Salvelinus fontinalis) following parental dietary exposure

AbstractThe toxicity of 2, 3, 7, 8‐tetrachlorodibenzo‐p‐dioxin (TCDD) to the early life stages of F1 generation brook trout (Salvelinus fontinalis) was determined, when dosed by maternal transfer. Effects were compared across six treatments, including a control. The experimental groups, based on TCDD concentrations in freshly spawned eggs (control, 41, 84, 156, 285, and 517 pg TCDD per gram egg [wet weight]), were established by treating 1.5‐year‐old brook trout with TCDD‐dosed food throughout the period beginning with final gonadal maturation and continuing until just prior to spawning. Measurement endpoints assessed at various times throughout the study, included (1) TCDD and lipid concentrations in ovaries, eggs, alevins, and juvenile fish, (2) mortality, (3) fertility indices, (4) growth, (5) juvenile sex ratios, (6) pathology, and (7) cytochrome P4501A1 mRNA (CYPIA1) levels in embryos. The cumulative lethality dose response was determined just prior to hatch, at swim‐up, and at the end of the study. The concentration in eggs causing 50% mortality (LCegg50) at swim‐up and at the end of the study were 138 and 127 pg TCDD per gram egg, respectively. No treatment effects were observed on fertility, growth, or juvenile sex ratios. Pathologies were observed that have been previously reported in TCDD‐exposed salmonid embryos, including edema in free embryos from all TCDD‐exposed treatment groups at hatch. At the end of the test, juvenile fish from all treatments exhibited an increased prevalence of exophthalmia compared to the controls. The ECegg50 for this effect was 117 pg TCDD per gram egg. Histological analysis of the end‐of‐test juveniles, in gonad, nerve, vertebrae, neuromasts, liver, kidney, gut, gas bladder, skin, and blood vessels, did not indicate any significant pathology below the LCegg50. P4501A1 mRNA levels in whole 91‐d postspawn free embryos from the 84 pg TCDD per gram egg treatment were 2.8 times higher compared with control or 41‐pg TCDD per gram egg treatments. Results from this study were consistent with other published results of TCDD effects on salmonids, in which the exposures occurred after fertilization.

17 beta-estradiol hydroxylation catalyzed by human cytochrome P450 1B1.

The 4-hydroxy metabolite of 17 beta-estradiol (E2) has been implicated in the carcinogenicity of this hormone. Previous studies showed that aryl hydrocarbon-receptor agonists induced a cytochrome P450 that catalyzed the 4-hydroxylation of E2. This activity was associated with human P450 1B1. To determine the relationship of the human P450 1B1 gene product and E2 4-hydroxylation, the protein was expressed in Saccharomyces cerevisiae. Microsomes from the transformed yeast catalyzed the 4- and 2-hydroxylation of E2 with Km values of 0.71 and 0.78 microM and turnover numbers of 1.39 and 0.27 nmol product min-1.nmol P450-1, respectively. Treatment of MCF-7 human breast cancer cells with the aryl hydrocarbon-receptor ligand indolo[3,2-b]carbazole resulted in a concentration-dependent increase in P450 1B1 and P450 1A1 mRNA levels, and caused increased rates of 2-, 4-, 6 alpha-, and 15 alpha-hydroxylation of E2. At an E2 concentration of 10 nM, the increased rates of 2- and 4-hydroxylation were approximately equal, emphasizing the significance of the low Km P450 1B1-component of E2 metabolism. These studies demonstrate that human P450 1B1 is a catalytically efficient E2 4-hydroxylase that is likely to participate in endocrine regulation and the toxicity of estrogens.

Expression of rat microsomal epoxide hydrolase during pregnancy

Microsomal epoxide hydrolase (mEH) protein and messenger ribonucleic acid (mRNA) levels were assessed in maternal rats during pregnancy and mEH gene expression was compared with cytochrome P450 expression. Immunoblot analysis using goat anti-rat mEH antibody showed that hepatic mEH levels in adult rats at day 12 of gestation were comparable to those in virgin female rats at the same age, whereas mEH protein levels were substantially decreased by ~70% at day 20 of pregnancy. Northern and slot blot analyses revealed that hepatic mEH mRNA levels failed to be modulated at day 12 of pregnancy, whereas mEH mRNA levels were decreased to ~20% of virgin control in late pregnancy ( i.e. day 20), which was consistent with the result of mEH immunoblot analysis. Hepatic cytochrome P4502E1 levels were also diminished at day 12 and day 20 of gestation by ~50% and ~70%, respectively, relative to virgin control rats, as supported by immunoblot analysis. Hepatic P4502E1 mRNA levels at day 12 and day 20 of pregnancy were also significantly decreased to 30% and 8% of virgin rats at 17 weeks of age, respectively, demonstrating that suppression in P4502E1 protein levels accompanied decreases in its mRNA expression. The levels of cytochrome P4501A2 mRNA at either day 12 or day 20 of pregnancy was decreased by ~50% relative to virgin female rats, which was less than that observed in mEH or P4502E1 expression. Pregnancy failed to affect P4501A1 mRNA levels, which were not detectable in female rats at 17 weeks of age. Renal mEH mRNA levels in maternal rats at day 20 of gestation were also decreased to 25% of virgin control. These results demonstrated that levels of mEH and P4502E1 proteins decreased significantly in late pregnancy with concomitant decreases in their mRNA levels.

Quantitative RNA-Polymerase Chain Reaction-DNA Analysis by Capillary Electrophoresis and Laser-Induced Fluorescence

Quantitative RNA-polymerase chain reaction (RNA-PCR) is an extremely powerful analytical tool owing to its specificity and high level of sensitivity. Quantitative RNA-PCR is, however, highly labor intensive. No analytical method currently exists that can accurately and rapidly quantitate the small quantities of DNA in RNA-PCR reaction mixtures. We have developed a method using capillary electrophoresis and laser-induced fluorescence to detect YOYO-1 complexes of DNA produced by PCR. RNA-PCR mixtures can be analyzed either directly (without primer and protein removal) or by electrokinetic injection following desalting. Modified competitive and multiplex competitive RNA-PCR assays for glyceraldehyde-3-phosphate dehydrogenase and P4501A1 were tested in a series of mixtures containing equal concentrations, but different proportions, of RNA from untreated (essentially no P4501A1 mRNA) and 2,3,7,8-tetrachlorodibenzo- p-dioxin-treated (high levels of P4501A1 mRNA) HepG2 cells. Twofold differences in concentrations between two P4501A1 mRNA solutions could be detected by competitive RNA-PCR. Glyceraldehyde-3-phosphate dehydrogenase concentrations were constant throughout. Multiplex competitive PCR produced more variable results due to the presence of contaminating peaks, which hindered accurate area integration. These data demonstrate the potential usefulness of capillary electrophoresis in a variety of quantitative PCR applications.

Retinoic acid antagonizes basal as well as coal tar and glucocorticoid-induced cytochrome P4501A1 expression in human skin.

Cytochrome P4501A1 is known to be expressed in skin and thus has been implicated in the pathogenesis of skin cancer due to certain environmental carcinogens. Retinoic acid (RA) has been used in chemoprevention of certain skin and other epithelial cancers. Therefore, we used Northern and Western analysis to determine the effect of externally applied RA on basal P4501A1 expression. RA reduced basal levels of P4501A1 mRNA and protein by 68 (n = 14, P = 0.005) and 75% (n = 7, P = 0.04) respectively. RA application also reduced basal levels of P4501A2 (another P4501A1 subfamily member) mRNA by 93% (n = 7, P = 0.001) as determined by reverse transcription-polymerase chain reaction. Interestingly, P4501A1 mRNA expression induced by coal tar or glucocorticoid (clobetasol) was reduced 46 (n = 10, P = 0.003) and 69% (n = 5, P < 0.05) respectively by RA co-application. Downregulation of basal P4501A1 expression and antagonism of coal tar mediated P4501A1 induction by RA may be a mechanism involved in chemo-prevention of skin and other epithelial cancer by RA.

Acetaminophen Toxicity in Cultured Trout Liver Cells: II. Maintenance of Cytochrome P450 1A1

Acetaminophen was demonstrated to maintain cytochrome P450 1A1 (P450 1A1) in isolated rainbow trout liver cells cultured in serum-free medium. This novel finding was characterized in detail. Cultured trout liver cells retained their ability to respond to typical 1A1 inducers in vitro; induction of ethoxyresorufin O-deethylase (EROD) activity was readily demonstrated by exposing liver cells from control trout to β-naphthoflavone (BNF), Aroclor 1254, or 7,12-dimethylbenz[a]anthracene. BNF was the most potent inducer studied and was used in further experiments. High levels of EROD activity, immunoreactive 1A1, and 1A1 mRNA were expressed in liver cells prepared from trout pretreated with BNF. However, all of these 1A1-specific indicators rapidly declined when cells from BNF-treated trout were placed in culture, and BNF in culture medium was not effective in maintaining EROD activity. Immunohistochemical studies suggested that addition of acetaminophen to liver cells prepared from BNF-induced trout helped maintain elevated levels of P450 1A1. Total cytochrome P450, EROD activity and immunoreactive P450 1A1 were retained in liver cells from BNF-induced trout by the addition of acetaminophen, in a dose-dependent manner. The concentrations of acetaminophen most effective in maintaining P450 1A1 produced cytotoxic effects, including vesiculation of endoplasmic reticulum. Furthermore, the acetaminophen maintenance of P450 1A1 was primarily attributed to elevated levels of P450 1A1 mRNA. In contrast to BNF, acetaminophen was not capable of inducing 1A1 in liver cells prepared from control trout. This is the first report to demonstrate that acetaminophen can help maintain P450 1A1 and that this effect is exerted at the level of P450 1A1 mRNA.

Transient induction of cytochrome P450 1A1 mRNA by culture medium component in primary cultures of adult rat hepatocytes.

Because the metabolic environment can alter gene expression in cultured cells, we examined the effects of change of medium on the levels of several cytochrome P450 mRNAs in primary cultures of rat hepatocytes maintained on Matrigel. The amounts of P450 1A2, 2B1/2, or 3A1 mRNA were unaffected by changing the medium. In contrast, P450 1A1 mRNA levels were increased 1 to 2 h after media change, reached maximum levels by 6 h, and declined to baseline by 24 h. Supplementing day-old media with components of the medium revealed that only addition of amino acids resulted in 1A1 mRNA induction. From the results of direct additions and omissions, we showed that tryptophan, but not histidine, was largely responsible for the 1A1 mRNA induction. Moreover, mild photoactivation of the tryptophan resulted in a substantially increased magnitude of 1A1 mRNA induction. The time course for 1A1 mRNA induction by treatment with photoactivated tryptophan was identical to that observed after medium change. Treatment of hepatocyte cultures with beta-naphthoflavone, which is metabolized by 1A1, also resulted in a transient 1A1 mRNA induction time-course profile over a 24-h period, whereas treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin, which is relatively stable to metabolic transformation, produced sustained elevations of 1A1 mRNA, suggesting that the transient response to tryptophan also may involve metabolism of the inducer. Our results extend previous data showing that oxidized products of tryptophan induce 1A1, and suggest that the transient nature of the induction may be due to elimination of the activated tryptophan molecule.

Effects of cycloheximide on the induction of CYP1A1 gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in three human breast cancer cell lines.

Treatment of MCF-7, MDA-MB-231 and Hs578-T human breast cancer cell lines with 10(-9) M 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1A1 gene expression in the MCF-7 but not in the MDA-MB-231 or Hs578-T cells. Pretreatment of the cells with 10(-5) M cycloheximide results in significantly increased P4501A1 mRNA levels in all three cells lines. However, in cells co-treated with 10(-5) M cycloheximide plus 10(-9) M TCDD, an induced response by TCDD was observed in the MCF-7 and MDA-MB-231 but not in Hs578-T cells. Gel-retardation assays of nuclear extracts from the three cell lines complexed with a 32P-labeled dioxin-responsive element (DRE) gave a TCDD-inducible retarded band only in the MCF-7 and MDA-MB-231 cells. A retarded band with a similar mobility was observed in nuclear extracts from Hs578-T cells treated with either 10(-9) M TCDD or DMSO (solvent control). These results suggest that aryl hydrocarbon non-responsive MDA-MB-231 and Hs578-T human breast cancer cell lines contain the CYP1A1 gene and treatment with cycloheximide increases both constitutive and TCDD-induced CYP1A1 gene expression.

Characterization of the rabbit CYP1A1 and CYP1A2 genes: Developmental and dioxin-inducible expression of rabbit liver P4501A1 and P4501A2

In adult rabbits, the CYP1A1 and CYP1A2 genes are expressed constitutively. Exposure to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) leads to elevations in both CYP1A1 and CYP1A2 gene products ( S. T. Okino et al., 1985, Proc. Natl. Acad. Sci. USA 82, 5310–5314 ). In this report, we have characterized the rabbit CYP1A1 and CYP1A2 genes, and analyzed the pattern of expression of these genes in neonatal animals following exposure to TCDD. Genomic clones encoding the entire rabbit CYP1A1 and CYP1A2 genes were characterized. Restriction enzyme analysis and partial DNA sequence analysis identified the seven exons for the CYP1A1 and CYP1A2 genes. Primer extension analysis using mRNA from TCDD-treated neonatal rabbits helped confirm the start of transcription for the CYP1A genes. The length of the noncoding first exon of the CYP1A1 gene was 74 bases, compared to 90 and 88 bases for the human and rodent CYP1A1 genes. The length of the noncoding CYP1A2 gene first exon was 53 bases, similar to its counterpart in human and rodents. DNA sequence analysis of the 5′ regulatory regions and comparison to the rodent and human CYP1 genes demonstrated that the rabbit CYP1A1 and CYP1A2 genes were most similar to their human orthologs. The 5′ region of the CYP1A1 gene contained several consensus dioxin (Ah)-receptor responsive elements (XREs), while no functional XRE sequences were identified in the CYP1A2 gene. When expression of the two genes were monitored, a small amount of constitutive P4501A1 mRNA was detected in neonatal rabbits from the ages of 1 to 17 days, while P4501A2 mRNA levels could not be observed until 8–12 days postpartum. In response to TCDD treatment, P4501A1 mRNA levels were inducible at all neonatal time points, while P4501A2 mRNA levels could not be induced until the animals were 3–5 days postpartum. While the dioxin Ah-receptor most likely plays a major role in the induction of these genes by TCDD, early expression of the CYP1A1 and CYP1A2 genes is differentially regulated in a developmental fashion.

The mechanism of action of α-naphthoflavone as an inhibitor of 2,3,7,8-tetrachlorodibenzo- p-dioxin-induced CYP1A1 gene expression

Treatment of rat hepatoma H-4-II E cells with α-naphthoflavone (αNF) (10 −8, 10 −7, 10 −6 m) resulted in only minimum induction of ethoxyresorufin O-deethylase (EROD) activity and cytochrome P4501A1 mRNA levels only at 10 −6 m. In contrast, 2,3,7,8-tetrachlorodi-benzo- p-dioxin (TCDD) caused maximum or near maximum induction responses at 10 −8 and 10 −9 m. In a timecourse study with TCDD (10 −9 m), and TCDD plus αNF (cotreated), αNF significantly inhibited the induction of EROD activity and cytochrome P4501A1 mRNA levels by TCDD for 6–24 h after initial exposure of the cells to the chemicals. In addition, treatment of the cells with 10 −9 m TCDD in the presence or absence of 10 −8, 10 −7, and 10 −9 m αNF showed that the latter compound inhibited the induction effects by TCDD in a concentration-dependent manner and these inhibitory effects could be overcome, in part, by a higher concentration of TCDD (10 −8 m). Treatment of the rat hepatoma H-4-II E cells with [ 3H]TCDD showed that within 60 min, there was an initial rapid increase in nuclear [ 3H]TCDD receptor complex levels (38 fmol/mg protein) which decreased to less than 10 fmol/mg protein within 4 h and remained relatively constant for up to 24 h. However, in cells treated with [ 3H]TCDD (10 −9 m) plus αNF (10 −6 m) the levels of the nuclear [ 3H]TCDD receptor complex were <5 fmol/mg protein throughout the 24-h time course. These data, coupled with the results which indicate that the αNF competitively inhibits the binding of [ 3H]-TCDD to the cytosolic aryl hydrocarbon (Ah) receptor, suggest that αNF inhibits the TCDD-mediated induction of CYP1A1 gene transcription and translation by direct competition for cytosolic Ah receptor binding sites.