Michael Steinberg

Induction of Cyp1a-1 and Cyp1a-2 gene expression by a reconstituted mixture of polynuclear aromatic hydrocarbons in B6C3F1 mice

The potential non-additive interactions of polynuclear aromatic hydrocarbon (PAH) mixtures as inducers of Cyp1a-1 and Cyp1a-2 gene expression were investigated in B6C3F1 mice using a reconstituted PAH mixture. The chemical composition (% by weight) of the reconstituted PAH mixture was: 2-ring PAHs--indan (0.22), naphthalene (23.8), 2-methylnaphthalene (23.2) and 1-methylnaphthalene (13.3); 3-ring PAHs--acenaphthylene (7.7), acenaphthene (0.6), dibenzofuran (0.7), fluorene (4.3), phenanthrene (10.5) and anthracene (3.4); > or = 4-ring PAHs--fluoranthene (2.4), pyrene (4.3), benz[a]anthracene (1.4), chrysene (1.5), benzo[b]fluoranthene (0.8), benzo[k]fluoranthene (0.9) and benzo[a]pyrene (0.9). The composition of the 2-, 3- and > or = 4-ring PAH fractions were based on the relative concentration of individual PAHs as noted above. The > or = 4-ring PAH fractions were based on the relative concentration of individual PAHs as noted above. The > or = 4-ring PAH fraction and reconstituted mixture induced hepatic microsomal ethoxyresorufin O-deethylase (EROD) activity and Cyp1a-1 mRNA levels, whereas the 2- and 3-ring PAHs were only weakly active. Direct comparison of the potencies of the reconstituted mixture and > or = 4-ring PAHs showed that the Cyp1a-1 induction activity of the reconstituted mixture was due to the > or = 4-ring PAHs. The reconstituted PAH mixture and > or = 4-ring PAHs also induced Cyp1a-2 hepatic mRNA levels and microsomal methoxyresorufin O-deethylase (MROD) activity; however, their dose-response curves indicated that the reconstituted PAH mixture was more potent as a Cyp1a-2 inducer than the > or = 4 ring PAHs. The differences in potency were due to 3-ring PAHs which were found to be strong inducers of hepatic Cyp1a-2 mRNA levels and microsomal MROD activity at the lowest dose administered (37 mg/kg). The 3-ring mixture caused a maximal 29-fold increase in hepatic MROD activity at a dose of 292 mg/kg, but only 28% of maximal induction of EROD activity. Northern analysis of liver mRNA from mice treated with 3-ring PAHs showed that there was minimal induction of Cyp1a-1 mRNA levels. The 3-ring PAHs did not competitively bind to the mouse hepatic cytosolic aryl hydrocarbon (Ah) receptor suggesting that 3-ring PAHs are a new class of Cyp1a-2 inducers which do not act through the Ah receptor.

Exposure of killifish to benzo[a]pyrene: comparative metabolism, DNA adduct formation and aryl hydrocarbon (Ah) receptor agonist activities

Benzo[a]pyrene (BaP) elicited a dose-response induction of hepatic CYP1A1 gene expression in killifish characterized by increased CYP1A1 mRNA levels, ethoxyresorufin O-deethylase (EROD) and aryl hydrocarbon hydroxylase (AHH) activities. There were marked differences in the maximally-induced EROD (1529 pmol mg−1min−1) and AHH (377 pmol mg−1 min−1) activities and these differences were observed at all ume points and at concentrations of BaP from 1 to 50 mg/kg. Treatment of killifish with BaP did not significantly affect binding of [125I]epidermal growth factor (EGF) to hepatic plasma membranes containing the EGF receptor. There was a dose-dependent increase in formation of DNA adducts in killifish treated with 1-50 mg/kg of BaP. Two-dimensional thin-layer chromatographic analysis of the [32P] labeled adducted nucleoudes revealed one major spot. In a time course study over a period of 2–14 days, the relative DNA adduct levels in fish treated with 5 mg/kg BaP were constant. The most sensitive indicator of exposure to BaP was the dose-dependent formation of biliary metabolites in which there was a > 40-fold increase (compared to untreated animals) in fluorescent metabolites formed at the lowest dose of BaP (1 mg/kg) used in this study. Killifish expressed relatively high levels (203 fmol/mg) of the nuclear aryl hydrocarbon (Ah) receptor which was isolated from nuclear extracts of fish treated with [3H]-2,3,7,8-tetrachlorodibenzo p-dioxin. The nuclear Ah receptor complex sedimented at 6.0 S which was similar to that observed in other animal species. Based on their CYPIA1 induction response to BaP and Ah receptor levels, the results of this study indicate that killifish are highly Ah-responsive.

Atypical cytochrome P450 induction profiles in glomerular mesangial cells at the mRNA and enzyme level: Evidence for CYP1A1 and CYP1B1 expression and their involvement in benzo[a]pyrene metabolism

Recent studies in this laboratory have shown that benzo[a]pyrene (BaP) modulates growth factor-related gene expression and proliferation of renal glomerular mesangial cells (GMCs) in vitro. Because many of the toxic and biochemical effects of this polycyclic aromatic hydrocarbon are mediated through oxidative metabolism, the present studies were conducted to examine the patterns of cytochrome P450IA1 (CYP1A1) and P4501B1 (CYP1B1) inducibility in mesangial cells and the molecular consequences of this response. Exposure of cultured GMCs to BaP (30 microM) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 10 nM) for 24 hr induced CYP1A1 mRNA levels, a response abolished by cotreatment with 10 microM cycloheximide. The pattern of hydrocarbon inducibility was atypical in that BaP was a more effective inducer of CYP1A1 gene expression than TCDD, and both hydrocarbons induced aryl hydrocarbon hydroxylase (AHH) activity, but not ethoxyresorufin-O-deethylase activity. Cotreatment with alpha-naphthoflavone (alpha NF, 1 microM) or ellipticine (ELLIP, 0.1 nM) only partially inhibited the induction of AHH activity by BaP (30 microM). BaP and TCDD also induced expression of the CYP1B1 protein and the pattern of induction was comparable to that observed for CYP1A1. Treatment of GMCs with 30 microM BaP was associated with the formation of eight DNA adducts, and their occurrence could be inhibited by pretreatment with alpha NF (1 microM), but not ELLIP (0.1 nM). These results demonstrate that CYP1A1 and CYP1B1-related activities are induced in GMCs by BaP and TCDD and this induction is associated with metabolism of BaP to reactive intermediates that bind covalently to DNA.

Immunotoxicity of a reconstituted polynuclear aromatic hydrocarbon mixture in B6C3F1 mice

Previous studies on the immunotoxicity of a complex mixture of polynuclear aromatic hydrocarbon (PAH) by-products from a manufactured gas plant indicated possible synergistic interactions which were investigated by determining the immunosuppressive effects of a reconstituted PAH mixture in female B6C3F1 mice challenged with TNP-haptenated sheep red blood cells (SRBCs) (T-cell-dependent) or trinitrophenyl-lipopolysaccharide (TNP-LPS) (T-cell-independent) antigens. The reconstituted PAH mixture contained the following 17 congeners: 2-rings (indan, naphthalene, 1- and 2-methylnaphthalene), 3-rings (acenaphthylene, acenaphthene, dibenzofuran, fluorene, phenanthrene and anthracene), and > or = 4-rings (pyrene, fluoranthene, benz[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene and benzo[a]pyrene), and resembled mixtures identified as by-products from manufactured gas plants. The reconstituted mixture and the 2-, 3- and > or = 4-ring PAH fractions all caused a dose-dependent decrease in the splenic plaque-forming cell (PFC) response to SRBCs or TNP-LPS, and their ED50 values for the four treatment groups were 86, 354, 145, and 23 or 163, 439, 637 and 31 mg/kg, respectively. The corresponding ED50 values for decreased serum anti-TNP IgM levels for these same mixtures were (TNP-haptenated SRBCs, T-cell-dependent) 144, 231, 42 and 27 units, respectively, and (TNP-LPS, T-cell-independent) 161, 406, 312 and 69 units, respectively. The suppression of anti-TNP IgM titers was similar to the suppression of the PFC response and shows that antigen-specific immunoglobulin titer can be used as a biomarker of PAH exposure. A direct comparison of the immunotoxic responses of the reconstituted PAH mixture and the corresponding dose of the > or = 4-ring PAHs indicated that the latter fraction was primarily responsible for the activity of the reconstituted mixture.

An enzyme-linked immunosorbant assay (ELISA) specific for antibodies to TNP-LPS detects alterations in serum immunoglobulins and isotype switching in C57BL/6 and DBA/2 mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds

Abstract An enzyme-linked immunosorbant assay (ELISA) was developed to detect IgM and IgG antibodies specific for trinitrophenyl-lipopolysaccharide (TNP-LPS). Treatment of C57BL/6 and DBA/2 mice with 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) and other aryl hydrocarbon (Ah) receptor agonists followed by immunization with TNP-LPS resulted in a dose-dependent decrease in serum IgM which paralleled the decrease in the splenic PFC response. The ED 50 values for the IgM and splenic PFCs in C57BL/6 mice for 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD), 3,3′,4,4′,5-pentachlorobiphenyl (pentaCB) and 3,3′,4,4′,5,5′-hexaCB were 2.8 and 1.6, 11 and 14, and 25 and 20 μg/kg, respectively; in the less Ah-responsive DBA/2 mice, the ED 50 values were 8.5 and 10, 61 and 69, and 73 and 71 μg/kg, respectively. In addition, treatment of C57BL/6 mice with TCDD resulted in alterations of serum IgG relative to IgM and a delay of isotype switching was observed after immunization and boosting with TNP-LPS. This ELISA may prove to be a useful tool in monitoring immune function during long-term exposure of mice to TCDD and related compounds and exploring the mechanism of Ah receptor-mediated immunosuppression.

Isolation and characterization of variant benzo[a]pyrene-resistant T47D human breast-cancer cells

T47D human breast cancer cells were grown in I μM benzo[a]pyrene (BaP) for 3.5 months, and 2 BaP‐resistant (BaPR) variant cell lines (C5 and C10) were isolated. Decreased aryl hydrocarbon (Ah)‐responsiveness in the C5 and C10 BaPR cells was characterized by lower (80 to 90%) induction of CYPIAI‐dependent activity by 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD), lower levels of the nuclear Ah receptor complex and significantly decreased Ah receptor mRNA levels. Nuclear estrogen receptor (ER) binding and ER mRNA levels were similar in wild‐type and mutant cell lines, whereas epidermal growth factor receptor mRNA levels were significantly decreased in the variant BaPR T47D cells. 17β‐Estradiol induced proliferation of both wild‐type and BaPR T47D cells, and TCDD inhibited this response but did not down‐regulate nuclear ER levels. The unique characteristics of the BaPR T47D variant cells will be used to further elucidate the mechanism of interaction between the ER and Ah receptor signalling pathways.

Induction of Transient Cell Proliferation by Manufactured Gas Plant Residue in B6C3F1 Male Mice

Abstract Fifteen day old B6C3F1 male mice were exposed by intraperitoneal (IP) injection to a Maximally Tolerated Dose of Manufactured Gas Plant (MGP) residue (1:32 dilution in DMSO or 1:8 in corn oil), 375 μg total of benzo[a]pyrene in DMSO or corn oil (CO) or DMSO and CO vehicle alone. On days 2, 4 and 7 post-treatment mice were injected IP with bromodeoxyuridine (BRdU) at a concentration of 50 μg/g of weight 1 hour prior to euthanasia. Immunoperoxidase staining for BRdU was performed and mitotic index (MI) determined for target tissues: bronchial mucosa, forestomach and liver. In MGP-exposed mice, MI in bronchial mucosa was increased 842% with DMSO vehicle and 555% in CO vehicle at day 2, MI was increased in forestomach mucosa by 40% at day 4 and MI was increased in liver by 50% at day 7 compared to controls. Therefore, complex mixtures such as MGP residue are capable of inducing transient cell proliferation. (Supported by EPRI RP2963-04)

Validation of Bioassays for Assessing the Contamination of Marine Environments

Abstract Various biomarkers were used to determine the exposure of fish (Arius felis and Micropogon undulatus) from Galveston Bay (GB), Texas, USA to organic contaminants. Sediment levels of polynuclear aromatic hydrocarbons (PAHs) in GB ranged from 81 to >1000 ng/g and polychlorinated biphenyls (PCBs) were <20 ng/g at all stations. No significant differences in hepatic concentrations of contaminants and ethoxyresorufin O–deethylase (EROD) activity, CYPIA mRNA levels, and DNA adducts were found in A. felis from GB. However, significant differences in biliary concentrations of naphthalene, phenanthrene, and benzo[a]pyrene metabolites were observed. Induced EROD activities and elevated levels of biliary PAH metabolites were measured in M. undulatus from the two most contaminated sites in GB. Induction toxic equivalents (I-TEQs), derived from dosing rat hepatoma H4IIE cells with hepatic extracts of A. felis, were correlated with tissue levels of ≥4–ring PAHs.

Enhanced Activity of Polynuclear Aromatic Hydrocarbon Mixtures as aryl Hydrocarbon (Ah) Receptor Agonists

Abstract The relative potencies of benzo[a]pyrene and a complex mixture of polycyclic aromatic hydrocarbons (PAH) produced as by-products of manufactured gas plant (MGP) residues as inducers of hepatic microsomal ethoxyresorufin O-deethylase (EROD) were determined in the B6C3F1 mouse. The ED50 values for the induction response were 78 and 65 mg/kg for benzo[a]pyrene and the MGP-PAH mixture, respectively, although benzo[a]pyrene and other compounds containing four or more rings were only trace components of this mixture. A comparison of the EROD induction potencies of benzo[a]pyrene and the MGP-PAH mixture showed that the mixture was approximately 706 times more active than expected based on its benzo[a]pyrene content (0.17%). The enhanced activity of the MGP-PAH mixture was also observed for several other aryl hydrocarbon (Ah) receptor-mediated responses, including inhibition of the splenic plaque-forming cell (PFC) response to both T-cell-dependent and independent antigens in B6C3F1 mice. The nature of t...