Induction Of Cytochrome P450IA1 Research Articles

Benzo(a)pyrene-Induced Alterations in Growth-Related Gene Expression and Signaling in Precision-Cut Adult Rat Liver and Kidney Slices

Benzo(a)pyrene (BaP) and related aromatic hydrocarbons are suspected carcinogens; however, the molecular basis underlying tumorigenesis remains unclear. To identify acute molecular targets of BaP within the liver and kidney, precision-cut slices harvested from naive, adult female Sprague–Dawley rats were challenged with BaP (0.3–30 μM) for 0.5 to 24 h. BaP did not elicit cytotoxicity, as assessed by intracellular K+and ATP content and histological evaluation over the 24-h period. To determine if molecular signaling pathways were maintained in precision-cut slices, induction of the aryl hydrocarbon receptor (AhR) pathway was assessed following BaP challenge. Induction of cytochrome P450IA1 (P450IA1) mRNA and protein expression was observed in both liver and kidney slices. c-fosand c-Ha-rasgene expression was enhanced in liver, but not kidney, slices by BaP. c-junmRNA levels were decreased in liver and kidney slices, although the effect was earlier (0.5 h) in liver slices compared to kidney slices. BaP increased the DNA binding of nuclear proteins to the AP-1 consensus recognition element in liver, but decreased DNA binding in kidney slices. In contrast, DNA binding of NF-κB was not affected by BaP in either liver or kidney slices. These results suggest that acute BaP challenge is associated with altered expression of several growth-related genes and AP-1 signaling and establish precision-cut slices as a usefulin vitrosystem to investigate the molecular basis of BaP-induced tumorigenesis, including organ-specific differences.

Different In Vitro Systems Affect CYPIA1 Activity in Response to 2,3,7,8-Tetrachlorodibenzo- p-dioxin

The induction of cytochrome P450IA1 (CYP1A1) activity in human hepatoma cell lines has been used as a model response for exposure to the environmental contaminant, 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). CYP1A1 induction is mediated by the intracellular Ah receptor. We have designed a cell culture analogue system to evaluate the effect of low dose continuous exposures to TCDD such that the responses can be compared with traditional static in vitro exposures. The two-compartment model is designed to mimic aspects of time-dependent dose exposure in humans and consists of a ‘liver’ compartment with HepG2 cells and an ‘other tissues’ compartment. Exposure of microcarrier-attached HepG2 cells in spinner flask to a single dose of TCDD resulted in an EC 50 for ethoxyresorufin- o-deethylase (EROD) activity induction of 1.49 n m. Using a one-compartment reactor with continuous TCDD dosing resulted in an EC 50 for EROD induction of 0.69 n m TCDD. Finally, using a two-compartment reactor with continuous TCDD dosing resulted in an EC 50 for EROD induction of about 0.05 n m. Parallel studies using 3H-TCDD were performed to determine the amount of cell associated 3H-TCDD and subsequently to estimate the amount of bound Ah receptor. CYPIA1 mediated EROD activities in both cell lines correlated with the estimated amount of bound Ah receptor. To illustrate how these systems might alter estimates of risk, the data were evaluated to estimate the TCDD level which would increase CYPIA1 mediated EROD activity to 0.01% of the maximal response. The two-compartment reactor data yielded an estimate of allowable TCDD exposure of 1×10 −5 n m for an acceptable ‘risk’ level of 0.01%. In contrast, values were estimated as 4×10 −4 n m from the one compartment reactor and 4×10 −3 n m from spinner flasks. This work demonstrates the importance of design of the in vitro system on risk assessment.

Detection of CYP1A1 mRNA levels and CYP1A1Msp 1 polymorphisms as possible biomarkers of exposure and susceptibility in smokers and non-smokers

It is important to determine sensitive biomarkers for both exposure and susceptibility since differences in individual susceptibility to potentially hazardous chemicals may represent a major variable in the assessment of risk. Induction of cytochrome P450IA1 (CYP1A1) may be a measure of environmental exposure to aromatic hydrocarbons in cigarette smoke. This study investigated the use of reverse transcriptase-polymerase chain reaction (RT-PCR) to detect constitutive levels of CYP1A1 mRNA in the peripheral lymphocytes of a population of smokers and non-smokers as a potential marker of exposure. In addition, the presence of an Msp 1 restriction fragment length polymorphism was analyzed using a simple PCR method as a biomarker for susceptibility. DNA and RNA were isolated from the peripheral lymphocytes of 20 smokers and a matched group of non-smokers. RT-PCR was used to detect the endogenous levels of CYP1A1 mRNA with glyceraldehyde-3-phosphate dehydrogenase as a control gene. The 3'-region of CYP1A1 gene was amplified by PCR and underwent restriction digestion with Msp 1 to detect the polymorphism. The endogenous CYP1A1 expression as detected by RT-PCR was very low and variable and there was a slight but not significant increase in the smokers by comparison with non-smokers. Thirty-two of the volunteers were homozygous for the normal allele while 8 were heterozygous for the uncommon Msp 1 allele and none was homozygous for the polymorphism. The allele frequency (0.1) was found to be in Hardy-Weinberg equilibrium. Since only a slight increase was seen in endogenous CYP1A1 mRNA levels in the peripheral lymphocytes of smokers by comparison with non-smokers, the effect may have been diluted by variation in sensitivity to dose, a threshold of exposure effect, or the return of mRNA to baseline between exposures. The wide variation in mRNA levels may reflect the influence and exposure of different environmental factors. The sensitivity of PCR-based methods suggests that they may have an important role in future overall biomonitoring of exposure and susceptibility to environmental chemicals.

Possible Involvement of the AH Receptor in the Induction of Cytochrome P-450IA1 Under Conditions of Hydrodynamic Shear in Microcarrier-Attached Hepatoma Cell Lines

The exposure of two hepatoma cell lines, Hep G2 and Hepa-1, to moderate hydrodynamic shear, in microcarrier-attached suspension cultures, resulted in the transient induction of cytochrome P-450IA1 (CYP1A1). Both cell lines have been characterized with respect to their Ah receptor (AhR) concentrations and induce CYP1A1 in response to exposure to xenobiotics such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Using an AhR antagonist, α-naphthoflavone (α-NF) and a protein kinase C (PKC) inhibitor, staurosporine (ST), in the Hep G2 cell line, the induced CYP1A1 activity was modulated in the same manner as when the cells were coexposed to TCDD and either α-NF or ST. Exposure of the Hep G2 cell line to TCDD and shear resulted in both enhancement of the induced CYP1A1 activity in addition to a competitive response. Finally, using the wild type and AhR defective Hepa-1 cell lines, it was demonstrated that a functional AhR was required for shear-induced CYP1A1 expression. The data obtained in the three cell lines indicate a role for the AhR in the induction of CYP1A1 by shear in agitated microcarrier cultures.

Dioxin-receptor ligands in urban air and vehicle exhaust.

The ability of extracts of urban air and vehicle exhaust particulates to bind to the dioxin receptor has been determined. It was shown that such extracts do contain significant amounts of dioxin-receptor binding activity. The level of dioxin-receptor binding found in ambient air reflects its pollution level as determined by mutagenic activity. Furthermore, it was shown that the extracts of both urban air and vehicle exhaust particulates could provoke the induction of cytochrome P450IA1 in cultured rat hepatoma cells. Chemical fractionation of the extracts revealed that the majority of the dioxin-receptor binding activity from urban air and gasoline vehicle samples fractionated with the polycyclic aromatic compounds. However, unknown polycyclic aromatic compounds were responsible for the majority of the binding activity measured. In the case of diesel vehicle exhausts, the majority of the dioxin-receptor binding activity was found to be associated with nitro-polycyclic aromatic compounds. Studies with a variety of diesel fuels showed that the amount of dioxin-receptor ligands present in exhaust emissions are fuel-dependent and that substantial amounts of dioxin-receptor ligands are present in the semivolatile phase of exhaust emissions.

Peat induces cytochrome P450IA1 in Hepa-1 cell line. Comparison with fly ashes from combustion of peat, coal, heavy fuel oil and hazardous waste

When the effects of the products of various combustion processes - with coal, heavy fuel oil, peat or hazardous waste as a fuel - were compared, an anomalous correlation between the biologically (induction of cytochrome P450IA1 in the mouse hepatoma cell line Hepa-1) and the chemically estimated “TCDD equivalents” was observed. The inducing effect of the product from a peat combustion operation was stronger than that of the product from hazardous waste combustion, although the chemically estimated TCDD equivalents were clearly lower in the former one. A more detailed study of two different peat combustion processes showed that induction in one of the combustion processes was due to the “leakage” of unburned peat to the fly ash. In the other process, no leakage and no cytochrome P450IA1 induction was evident. This led us to a more detailed study of peat itself. One fraction of the peat extract was shown to be a very potent inducer of cytochrome P450IA1. However, only small amounts of polychlorinated dibenzodioxins (PCDDs) or furans (PCDFs) were present in this fraction. It is thus evident that there are cytochrome P450IA1 inducers in peat other than those revealed by the PCDD/PCDF analysis.

Induction of cytochrome P450IA1 and its recombinant construct in H4IIE rat hepatoma cells

1. 1. Our previous studies have shown that benzo(a)pyrene (BP), 3-methylcholanthrene (3MC) and tetrachlorodibenzofuran (TCDBF) can induce the expression of the cytochrome P450IA1 mRNA in the rat hepatoma cell line, H4IIE, although the kinetics of induction differed. 2. 2. In the present study, by using biochemical, immunochemical and recombinant DNA approaches, the effects of these inducers have been examined on the steady state level of endogenous cytochrome P450IA1 protein and on induction of chloramphenicol acetyltransferase activity (CAT) in the H4IIE cells transfected with pMC 1CAT (a recombinant construct consisting of CAT linked to 5' upstream DNA sequence of the rat cytochrome P450IA1 gene). 3. 3. From 7-ethoxyresorufin O-deethylase activity (EROD) and immunochemical analysis of cytochrome P450IA1, the optimal concentrations of BP, 3MC and TCDBF for induction in the H4IIE cells were determined as 1, 0.1-1 and 0.1 μ M, respectively. 4. 4. The elevated expression of the protein was more sustained in the TCDBF-exposed cells than in the BP or 3MC-treated cells. 5. 5. After 1.5 hr of treatment, little if any detectable P450IA1 protein was observed in the H4IIE cells although a considerable amount of mRNA was present. 6. 6. In addition, no cytochrome P450IA2 protein was detected in the control or induced H4IIE cells. 7. 7. H4IIE cells were transfected by pMC 1CAT, and the induction ratio of CAT expression in the transfected H4IIE cells after BP, 3MC or TCDBF treatment was 10-, 17- and 40-fold, respectively. 8. 8. These results indicate that the rat H4IIE cell line offers a valid homologous system for studies of the regulation of the rat cytochrome P450IA1 gene.

Regulation of 2,3,7,8-tetrachlorodibenzo- p-dioxin-inducible expression of aldehyde dehydrogenase in hepatoma cells

The environmental contaminant, 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) induces the expression of a number of genes. The biochemical process of the induction of aldehyde dehydrogenase (ALDH-3) was investigated in rat H4IIE hepatoma cells in culture. The kinetics of ALDH-3-induction exhibited parallel increases in the rate of transcription, mRNA, protein, and enzyme activity, all reaching a plateau at 36–48 h after addition of TCDD. Half maximal and maximal inductions occurred at 0.1 and 1 n m of TCDD, respectively. No significant changes in the half-life of ALDH-3 mRNA (14 h) were observed in the cells exposed to three different concentrations of TCDD. Other inducers of xenobiotic metabolism, such as 3-methylcholanthrene and β-naphthoflavone, also induced ALDH-3 mRNA to a similar level as TCDD, whereas antioxidants or electrophiles, such as tert-butylhydroquinone and dimethyl fumarate, did not show any induction of ALDH-3 mRNA. To examine the involvement of the aryl hydrocarbon receptor ( Ah receptor) in the induction of ALDH-3, mouse variant cell lines defective in cytochrome P450IA1-induction and a parental wild type cell line (Hepa1c1c7) were studied. ALDH-3 mRNA and the transcription of its gene were detected in TCDD-treated wild type cells, but not in the treated and untreated variant cells. These results demonstrate that TCDD induces transcription of the ALDH-3 gene via its binding to the Ah receptor.

Effects of inhibition and induction of cytochrome P-450 isozymes on hyperoxic lung injury in rats.

Pulmonary oxygen toxicity most likely results from excessive production of reactive oxygen species. The role of the cytochromes P-450 in this process is controversial because these enzymes have been reported both to enhance hyperoxic lung injury and to protect from the damaging effects of 100% oxygen. We sought to further determine the role of the cytochromes P-450 in hyperoxic lung injury by inhibiting and inducing pulmonary cytochrome P-450 isozymes in rats. Treatment with the cytochrome P-450 inhibitor cimetidine or 8-methoxypsoralen did not improve survival or reduce lung edema in rats exposed to 100% oxygen. The activity of cytochrome P-450IIB1, the major pulmonary cytochrome P-450 isozyme in rats, was clearly inhibited by 8-methoxypsoralen. beta-Naphthoflavone (beta NF), a selective inducer of cytochrome P-450IA1, was administered in two-dose and five-dose regimens. The two-dose regimen produced significant and sustained induction of cytochrome P-450IA1 activity, but survival in these rats was not improved when exposed to 100% oxygen. In rats treated with five doses of beta NF, a small increase in survival time was found from 71.1 +/- 8.7 to 88.0 +/- 20.2 h; however, there was no difference in the induction of cytochrome P-450IA1 activity between this five-dose regimen and the two-dose regimen. The small improvement in survival after five doses of beta NF is thus unrelated to cytochrome P-450IA1 induction. We conclude that neither inhibition of cytochrome P-450IIB1 activity nor induction of cytochrome P-450IA1 activity protects adult rats against hyperoxic lung injury.

Comparison of cytochrome P450IA1 induction with the chemical composition of fly ash from combustion of chlorine containing material

Polychlorinated dibenzo- p -dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) are ubiquitous in the environment. Combustion of organic material, particularly in the presence of chlorine, is a significant source of these harmful compounds. In this study, the induction of cytochrome P450IA1 - measured as aryl hydrocarbon hydroxylase and 7-ethoxyresorufin O-deethylase activities - in the mouse hepatoma cell line, Hepa-1, was used as a biological indicator of the presence of PCDDs and PCDFs in fly ash collected from combustion of chlorine containing material. The highest chlorine content in the combustions studied was 1.2 % (w/w) in a mixture of coal and bark. An amount corresponding to about 7.0 mg of the respective fly ash per ml of growth medium was enough to elicit a half-maximal (ED50) induction of cytochrome P450IA1 in Hepa-1 cells. The inducing compounds were located in the fly ash fraction which contained the PCDDs and PCDFs. There was a good correlation between the PCDD/PCDF composition of the fly ashes and the enzyme inducing effect they had.

The time-dependent increase in the binding of benzo[a]pyrene to DNA through (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide in primary rat hepatocyte cultures results from induction of cytochrome P450IA1 by benzo[a]pyrene treatment

The proportion and amount of benzo[a]pyrene (B[a]P) that binds to DNA through the carcinogenic (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide [(+)-anti-BPDE] increases with time of exposure to B[a]P in cell cultures derived from a number of species. Pretreatment of primary rat hepatocyte cultures for 12 h with 1 microgram B[a]P/ml medium increased the subsequent metabolism of [3H]B[a]P by 47% and [3H]B[a]P-DNA binding by 53% compared with acetone-pretreated hepatocytes. The amount of (+)-anti-BPDE bound to DNA in the B[a]P-pretreated hepatocytes increased 175%. B[a]P pretreatment also increased DNA-binding 2-fold in hepatocytes treated with [3H]7,8-dihydroxy-7,8-dihydro-B[a]P but had no effect on DNA binding in cells treated with anti-B[a]P-7,8-diol-9,10-epoxide. Western blotting showed that cytochrome P450IA1, which was not detectable prior to B[a]P treatment, was selectively increased by B[a]P treatment. A monoclonal antibody that specifically inhibits cytochrome P450IA1 reduced the binding of B[a]P to DNA by greater than 90% in microsomal preparations from B[a]P-pretreated hepatocytes. These results indicate that the time-dependent increase in the formation of (+)-anti-BPDE-DNA adducts results from an increase in the amount and proportion of B[a]P metabolized to this ultimate carcinogen by P450IA1 that is induced by the B[a]P treatment. The importance of P450IA1 induction by the B[a]P for its activation to this ultimate carcinogenic metabolite suggests that long-term exposure of cells to B[a]P could result in activation of a higher proportion of the B[a]P to the carcinogenic (+)-anti-BPDE.

Effect of dietary fiber on cytochrome P450IA1 induction in rat colonic mucosa.

The effect of dietary fiber on the induction of cytochrome P450IA1 in rat colonic mucosa after a single intragastric injection of 3-methylcholanthrene (3MC, 20 mg/kg) was investigated by examining the drug-metabolizing enzyme activity, immunoblotting for cytochrome P450IA1 and immunohistochemistry. 7-Ethoxycoumarin-O-deethylase activities were approximately 20-fold higher in microsomes from both proximal and distal portions of the colonic mucosa of control diet-fed 3MC-treated rats compared with those of control diet-fed untreated rats. Strong immunofluorescence for cytochrome P450IA1 was localized in the cytoplasm of the colonic mucosa surface epithelium from the control diet-fed 3MC-treated rats. 7-Ethoxycoumarin-O-deethylase activity and cytochrome P450IA1 content determined by immunoblotting were significantly lower in wheat bran-fed 3MC-treated rats than in control diet-fed 3MC-treated rats. Immunohistochemical analysis showed much weaker immunofluorescence for cytochrome P450IA1 in the surface epithelium of the colonic mucosa of the wheat bran-fed 3MC-treated rats. These observations suggested that dietary fiber can affect the induction of cytochrome P450IA1 in colonic mucosa by dietary inducers or carcinogens.

In situ pollution induced cytochrome P450 activity of freshwater fish: barbel ( Barbus barbus), chub ( Leuciscus cephalus) and nase ( Chondrostoma nasus)

The induction of cytochrome P450IA1 was detected in freshwater fish liver using 7-ethoxyresorufin- O-deethylase activity (EROD). Investigations took place in the river Durance in the south-east of France and revealed a strong induction downstream a chemical plant. This induction differed between the three species studied. The species Barbus barbus was more sensitive to the pollution. This might be due to the benthic feeding behaviour ofthat fish. The induction increased from February to October which was probably a concentration effect caused by the drastic drought that occurred in France in 1989.

Downregulation of the Ah receptor in mouse hepatoma cells treated in culture with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The aromatic hydrocarbon (Ah) receptor behaves as a ligand-dependent transcription factor in the induction of cytochrome P450IA1. In cells exposed to the Ah receptor ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the Ah receptor undergoes a transformation from a form with low affinity for nucleic acids (cytosolic receptor) into a form that preferentially associates with the cell nucleus (nuclear receptor). We followed the fate of the Ah receptor in mouse hepatoma cells during short-term exposure to [3H]TCDD by analyzing both cytosolic and nuclear fractions for specific binding. Nuclear Ah receptor levels increased over the first 2 h of treatment and then decreased to about 50% of maximal concentrations by 5 h after start of treatment. The decrease in nuclear receptor was not accompanied by a reappearance of detectable Ah receptor in the cytosolic fraction; further incubation with [3H]TCDD in cytosols from lysed cells did not label any additional receptor sites in cytosolic extract. By the 6th h of incubation, the total receptor population in the cell was only about 15-20% of that detected at the start of the incubation. The levels of specific binding detected were unaffected by up to 20 h of incubation with the vehicle DMSO, confirming that the presence of TCDD is required for the observed downregulation to occur. These results indicate that there is a substantial ligand-dependent loss in total Ah receptor during short-term exposure of cells to TCDD in culture.

Ah receptor mediating induction of cytochrome P450IA1 in a novel continuous human liver cell line (Mz-Hep-1): Detection by binding with [ 3H]2,3,7,8-tetrachlorodibenzo- p-dioxin and relationship to the activity of aryl hydrocarbon hydroxylase

The Ah receptor regulates induction of cytochrome P450IA1 and mediates certain toxicities of polyhalogenated aromatics such as 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). It has been characterized previously in continuous cell lines, notably the mouse hepatoma line Hepa 1, the human squamous cell carcinoma line A431, and the human liver cell line Hep G2. The present work extends our knowledge of the Ah receptor in continuous human liver cell lines. Ah receptor can be detected in Mz-Hep-1, a hepatitis B virus-negative cell line derived from a Thorotrast-induced hepatocellular carcinoma. The mean concentration of Ah receptor in Mz-Hep-1 cells was 341 ± 22 fmol/mg cytosol protein (mean ± SEM, nine separate determinations). This is equivalent to ~30,000 sites per cell. The concentration of Ah receptor in Mz-Hep-1 cells is similar to that in Hepa 1 cells and approximately three times higher than that in Hep G2 cells. The Mz-Hep-1 Ah receptor sedimented in continuous sucrose gradients at ~9 S. Specificity of binding by [ 3H]TCDD was demonstrated by competitive binding of non-radiolabeled 2,3,7,8-tetrachlorodibenzofuran, 3-methylcholanthrene (MC), and dibenz[ a, h]anthracene in 50-fold molar excess. Phenobarbital, which is not a substrate for P450IA1, did not compete with [ 3H]TCDD for binding to Mz-Hep-1 Ah receptor. Dexamethasone and estradiol also did not compete with [ 3H]TCDD for binding, suggesting non-identity of Ah receptor with glucocorticoid or estrogen receptor. In separate experiments, glucocorticoid receptor was identified in Mz-Hep-1 cells. By Scatchard plot analysis, the apparent equilibrium dissociation constant ( K d ) for binding of [ 3H]TCDD to Mz-Hep-1 Ah receptor was estimated to be 4.4 nM, compared to 0.8 nM in Hepa 1 cells. By Woolf plot analysis the K d was 5.4nM, compared to 1.2nM in Hepa 1 cells. The [ 3H]TCDD-Ah receptor complex extracted from nuclei of Mz-Hep-1 cells incubated with [ 3H]TCDD in culture at 37° sedimented at ~6 S under conditions of high ionic strength. Aryl hydrocarbon hydroxylase (AHH) activity was detectable in Mz-Hep-1 cells after pretreatment with inducing chemicals. Mz-Hep-1 cells have the highest concentrations of Ah receptor in any continuous human liver cell line thus far investigated. The Mz-Hep-1 Ah receptor is similar physicochemically to that described in murine systems. AHH activity is inducible in Mz-Hep-1 cells.

The formation of 3-methylcholanthrene-initiated lung tumors correlates with induction of cytochrome P450IA1 by the carcinogen in fetal but not adult mice : Miller MS, Jones AB, Park SS, Anderson LM. Perinatal Carcinogenesis Section, National Cancer Institute, Frederick, MD 21701-1013. Toxicol Appl Pharmacol 1990;104:235–245

The formation of 3-methylcholanthrene-initiated lung tumors correlates with induction of cytochrome P450IA1 by the carcinogen in fetal but not adult mice : Miller MS, Jones AB, Park SS, Anderson LM. Perinatal Carcinogenesis Section, National Cancer Institute, Frederick, MD 21701-1013. Toxicol Appl Pharmacol 1990;104:235–245

Induction of cytochrome P-450IA1 in fetal rat liver by a single dose of 3-methylcholanthrene

Pregnant Sprague-Dawley rats were treated with a single ip dose of either olive oil or 40 mg/kg of 3-methylcholanthrene on gestation day 20 and sacrificed at various times after injection. Determination of aryl hydrocarbon hydroxylase activity 24 hr after injection revealed that treatment with 3-methylcholanthrene resulted in a 10.5-fold stimulation of enzymatic activity in liver 800 × g supernatants. Western blot analysis with monoclonal antibody 1-7-1 confirmed these results and demonstrated the presence of a 3-methylcholanthrene-inducible P-450 isozyme. Using Northern and slot blot techniques, the induction of steady-state levels of CYPIA1 RNA was shown to occur as early as 4 hr following 3-methylcholanthrene injection. CYPIA1 RNA levels were induced 31.6-fold over values obtained from oil-treated tissues at this time. This appears to be the optimal time to study changes in the levels of CYPIA1 RNA gene expression in the fetus following transplacental exposure to polycyclic aromatic hydrocarbons. By 12 to 24 hr postinjection, the induction of CYPIA1 RNA levels declined to 3.5- to 8.5-fold above control values. These results demonstrate that the kinetics of induction of the CYPIA1 gene during the fetal period differed from that seen in adults.

Immunochemical detection of cytochrome P450IA1 induction in cod larvae and juveniles exposed to a water soluble fraction of North Sea crude oil

Using a flow-through biotest exposure system, cod eggs, larvae and juveniles were exposed to the water-soluble fraction (WSF) of North Sea (Statfjord B) crude oil at concentrations ranging from 40–300 μg l −1 (ppb) for 1–6 weeks. The WSF was analysed by GC/MS, and was shown to be dominated by low-aromatic compounds such as benzenes, toluene and xylenes (80–90%). Cytochrome P450IA1 levels were measured by immunochemical techniques in 10 000 × g supernatants of whole larvae or juvenile gill homogenates, and in juvenile liver microsomal fractions. With rabbit anti-cod P450IA1 IgG as primary antibody in an indirect ELISA, a clear induction response was observed in the exposed groups of both larvae and juveniles. The response was dose-dependent, and recovery in clean sea-water resulted in normalization of the induced P450IA1 levels. In larvae exposed during the egg stage, the induction process seemed restricted until the time of hatching. In juvenile cod, the response was observed in the gill in addition to the liver. The study demonstrates the usefulness of the cytochrome P450IA1 ELISA in detecting sublethal biological effects of pollutants in small sample sizes where enzyme activity is difficult to measure.

Induction of hepatic cytochrome P-450IA1 in pigeons treated in vivo with aroclor 1254 a commercial mixture of polychlorinated biphenyls (PCBS)

1. Treatment with a commercial mixture of polychlorinated biphenyls (PCBs) resulted in highly significant increases in pigeon hepatic microsomal proteins (100-fold), cytochrome P-450 (11-fold), cytochrome b 5 (7-fold), NADPH-cytochrome c-(P450) reductase (7-fold), ethoxycoumarin- O-deethylation (9-fold), aldrin epoxidase (22-fold), ethoxyresorufin- O-deethylation (48-fold), N-demethylation of dimethylnitrosamine (28-fold) but not of lauric acid 12-hydroxylation. 2. SDS-PAGE analysis of pigeon hepatic microsomal proteins induced by Aroclor 1254 suggested highly significant increases in the density of staining in bands of estimated M r, 51–52 kD, 54–54.5 kD, 57–58 kD, 59–60 kD and of 77.5–78.5 kD. 3. The induction of cytochrome P-450IA1 was confirmed by Western immunoblotting using the monoclonal antibodies MAB 1-12-3 and MAB 1-8-4. 4. There was agreement between the 8-fold increase in cytochrome P-450IA1 increased staining of microsomal proteins, as judged by SDS-PAGE, and the 24-fold increase in the amount of protein that reacted with the monoclonal antibodies MAB 1-12-3 and MAB 1-8-4, as judged by Western immuno-blotting. 5. It is concluded that treatment with a commercial PCB mixture resulted in the induction of several isoforms of pigeon hepatic cytochrome P-450 in a fashion that is likely to be similar to that reported for mammals.

Promotion of Mouse Lung Tumors by Bioaccumulated Polychlorinated Aromatic Hydrocarbons

Lung tumors initiated in infant Swiss mice by N-nitrosodimethylamine (NDMA) were promoted by a single dose of a mixture of polychlorinated biphenyls (PCBs), Aroclor 1254 (250 or 500 mg/kg) given 4 days later. The tumors were typical alveologenic adenomas, and their number increased gradually over the course of 1 year to a maximum 4-fold enhancement in average tumor number compared with those given NDMA alone. The time course pattern suggested continuous tumor stimulation by the nonmetabolized PCB congeners retained in the tissues. Content of the nine major bioretained PCB congeners in carcass, liver, and lung was determined at intervals after treatment. Several showed tissue-specific retention patterns: 2,3,3',4,4'-PCB was selectively retained in lung and liver, and 2,2',3,4,4', 5'-HCB in lung. In tests of the tumor-promoting ability of individual congeners, the 2,2',3,4,4',5'-HCB, an Ah receptor agonist, promoted lung tumors when given singly, whereas another prominent bioretained congener, the 2,2',4,4',5,5'-HCB, an Ah receptor antagonist, did not promote, and in fact abrogated the positive effect of the 2,2',3,4,4',5'-HCB. In a parallel examination of persistent biochemical effects, a single low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (5 nmole/kg) resulted in significant elevation of immunochemically detected protein and enzymatic activity of cytochrome P450 IA1 in lung for at least 12 weeks; these parameters were elevated for at least 30 weeks after a single dose of Aroclor 1254 (500 mg/kg). Taken together these results suggest that Ah-receptor-dependent induction of cytochrome P450 IA1 in mouse lung is correlated with and possibly causally involved in promotion of tumors by retained congeners.