Lewis V. Rodriguez

Synergistic activity of polynuclear aromatic hydrocarbon mixtures as aryl hydrocarbon (Ah) receptor agonists

The relative potencies of benzo[a]pyrene and a complex mixture of polynuclear aromatic hydrocarbons (PAHs) produced as by-products of manufactured gas plant (MGP) residues as inducers of hepatic microsomal ethoxyresorufin O-deethylase (EROD) activity 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. Analysis of the MGP-PAH mixture indicated that benzo[a]pyrene and other compounds containing four or more rings and which are known to induce EROD activity were only present as 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 potent than expected based on its benzo[a]pyrene content (0.17%). This induced P-450 activity could significantly increase the metabolism of the carcinogenic PAHs and thereby modulate the overall carcinogenicity of the mixture. The apparent synergistic activity of the MGP-PAH mixture was further investigated by comparing the activities of this mixture and benzo[a]pyrene for several other aryl hydrocarbon (Ah) receptor-mediated responses including (i) induction of hepatic CYP1A1 mRNA levels, (ii) transformation of the rat cytosolic Ah receptor to a complex which binds to a dioxin responsive element, (iii) induction of EROD activity and (iv) antiestrogenicity in MCF-7 human breast cancer cells, and (v) inhibition of the splenic plaque-forming cell (PFC) response to both T cell-dependent and independent antigens in B6C3F1 mice. For the EROD and CYP1A1 mRNA induction and cytosolic transformation activities and immunosuppressive effects, the MGP-PAH mixture was approximately 100-900 times more potent as an Ah receptor agonist than expected based on its benzo[a]pyrene content. The synergistic activity was lower (19-fold) for the antiestrogenic response in MCF-7 cells. The reason for the synergistic effects of the MGP-PAH mixture were not due to contamination of the mixture by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds and the results suggest that the enhanced potency of the mixture is due to unknown interactions between the individual PAHs present in the mixture.

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.

Heat-stress proteins of rat lung endothelial and mammary adenocarcinoma cells.

A rat mammary adenocarcinoma cell clone, MTC, and a rat lung endothelial cell clone, RLE cl.4, both syngeneic to the Fisher 344 rat, were compared for proteins synthesized at 37 degrees C and after a 1-h, 42 degrees C heat dose. The heat stress-induced or -enhanced synthesis of a series of molecular mass groups and isoelectric point species (isomers) was observed in both equilibrium and nonequilibrium two-dimensional gel electrophoresis. Tumor and endothelial cell heat-stress proteins (hsp) were strikingly similar with most hsp in 11 or 13 molecular mass groups having from 1 to 12 major isomers. In comparing the two cell types, 6 of about 23 major hsp isomers appeared different in equilibrium pH gels, with tumor cells seemingly exhibiting less synthesis of these 6 isomers. Four additional endothelial cell hsp isomers were apparent in nonequilibrium pH gels. Since two of these later hsp can be found at higher heat doses in tumor cells, some of these apparent differences between tumor and endothelial cells may be attributable to different dose ranges for induction of hsp. Fluorograms and silver-stained gels showed that several hsp were being synthesized at appreciable levels in unheated cells. However, there were hsp whose synthesis appeared to be de novo rather than representing enhanced synthesis of existing proteins. These last two observations were made in both tumor and normal cells. The constitutive levels of hsp synthesis appeared to be generally similar in unheated tumor and normal cells in vitro with few exceptions. These results indicate the presence of few unique hsp in syngeneic tumor and normal cells in vitro. However, focusing subsequent studies on the few differences may lead to insights concerning hyperthermic biology of tumor and normal cells, phenotypic differences between these cells, and roles of some hsp.

Toxic equivalency factor approach for risk assessment of combustion by‐products

Hazard and risk assessment of individual toxic chemicals utilizes data obtained from chronic toxicity and carcinogenicity studies in laboratory animals for regulating emission, cleanup or intake levels for individual chemicals. This approach can be utilized for problems associated with a single chemical emitted from a point source; however, in most situations, toxic chemicals are formed and emitted into the environment as complex mixtures of different structural classes of toxic/carcinogenic chemicals. Methodologies for risk assessment of complex mixtures have been developed for polychlorinated dibenzo‐p‐dioxins (PCDDs) and dibenzofurans (PCDFs) which are found as industrial and combustion by‐products. This methodology is based on the common mechanism of action for these chemicals and utilizes the most toxic PCDD/PCDF congener, 2, 3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD), as a reference compound. The relative potencies or toxic equivalency factors (TEFs) are assigned to the other relevant PCDD/PCDF congen...

Genetic Analysis of Nonhistone Chromosomal Protein Inheritance in Recombinant Inbred Mouse Strains Using Two-Dimensional Electrophoresis

Analysis of hepatic nonhistone chromosomal protein (NHCP) expression in male mice from progenitor strains (C3H/HeN, C57BL/6N), their F1 hybrid (B6C3), and seven recombinant inbred strains (RIs) (B6N×C3N) by high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) detected 16 NHCPs whose expression in RIs could be correlated to each other and with strain distribution patterns (SDP) of 20 genetic markers differing in the progenitors. Of the 400+ NHCP spots detected in RI 2D-PAGE maps, 172 were common to progenitors and all RIs. There was a characteristic absence of five NHCPs in one RI, Y. Ten C3H-specific and six C57-specific NHCP inherited in B6C3 also appeared in RIs. The SDP of C3H-specific NHCP 2 matched the SDP of beta-glucuronidase on chromosome 5 and carbonic anhydrase on chromosome 3, and C57-specific NHCP 5 SDP corresponded to that for nonagouti trait on chromosome 2. These 16 NHCP genetic marker inheritance differences detected in RIs add to the 23 previously established genetic marker differences between the progenitors.

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)

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...