Eric H. Weyand

Induction of CYP1A1 and CYP1A2 expressions by prototypic and atypical inducers in the human lung

The inducibility of cytochrome P4501A1 gene (CYP1A1) expression was examined in human lung samples from 27 subjects, using an explant culture system and semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. CYP1A1 transcripts were present in all of the lung specimens and were induced by the prototypic inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo[a]pyrene (B[a]P), and by the atypical inducers pyridine, nicotine, and omeprazole. 2-Hydroxypyridine was a better inducer than pyridine, implicating metabolites in CYP1A1 induction by the parent compound. The prototypical inducers were the most effective inducers in many samples but were ineffective in some samples in which the atypical compounds were effective inducers. Cytochrome P4501A2 (CYP1A2) transcripts were also detected in most of the lung specimens and were inducible in some specimens. The results show the suitability of the explant culture system for examining the inducibility of human pulmonary CYP1A1 and CYP1A2, indicate the heterogeneity in individual sensitivity to the induction, and underscore the need to include atypical inducers in studies of CYP1A inducibility in humans.

An investigation of the intradermal route as an effective means of immunization for microparticulate vaccine delivery systems

Among the common routes of parenteral immunization, the skin is the only site that can function as an immune organ. Skin-associated lymphoid tissue contains specialized cells that enhance the immune response. The intercellular space in the skin interstitium provides a connection to the lymphatic capillaries and vessels that terminate in peripheral immune organs like the lymph nodes and spleen. The potential of intradermal immunization with microparticulate vaccine delivery systems was investigated in this study. The microparticulates used were muramyl dipeptide (MDP) loaded ovalbumin microspheres (OVA-MSs) and fluorescent latex microspheres of fixed sizes of 2.3 and 2.1 microm diameter, respectively. Similar doses of OVA-MSs were injected subcutaneously (s.c.) and intradermally (i.d.) in mice. The induced OVA-specific IgG antibody immune response was found to be significantly higher in i.d. immunized mice as compared to those injected s.c. The sc and i.d. administration of fluorescent latex microspheres in mice demonstrated that the uptake and translocation of microspheres from the site of injection depends primarily upon the surface area of the microspheres. The enhancement in antibody production upon i.d. administration was explained on the basis of (i) an increased surface area of microspheres and a lower number of microspheres per injection site, and (ii) an increased probability of interaction with the immune cells of the skin. Efficient lymph node targeting observed from the id administered microspheres may be the result of both of these factors. The results of this study demonstrated that the intradermal route is an effective means of immunization for microparticulate vaccine delivery systems, requiring lower doses and resulting in a higher immune response as compared to the traditionally used sc route.

Exceptional tumor-initiating activity of 4-fluorobenzo[j]-fluoranthene on mouse skin : comparison with benzo[j]-fluoranthene, 10-fluoro-benzo[j]fluoranthene, benzo[a]pyrene, dibenzo[a,l]pyrene and 7,12-dimethylbenz[a]anthracene

Exceptional tumorigenic potency was observed with 4-fluorobenzo[j]fuoranthene (4-fluoroB[j]F) relative to benzo[j]fluoranthene (B[j]F) and 10-fluorobenzo[j]fluoranthene (10-fluoroB[j]F) in a mouse skin initiation promotion bioassay. Comparison of the tumorigenic response obtained at total initiating doses of 50, 100, and 1000 nmol firmly established the greater tumorigenic potency of 4-fluoroB[j]F. B[j]F produced a significant tumorigenic response only at total initiating doses of 100 and 1000 nmol per mouse. 10-FluoroB[j]F produced a significant tumorigenic response only at the highest initiating dose, 1000 nmol per mouse. In contrast, 4-fluoroB[j]F produced a significant tumorigenic response at all three doses. At a total initiating dose of 50 nmol, a 90% incidence of tumor-bearing mice with an average of 3.05 tumors per mouse was observed with 4-fluoroB[j]F. A second initiation promotion bioassay was performed to establish the tumorigenic potency of 4-fluoroB[j]F relative to benzo[a]-pyrene (B[a]P), 7,12-dimethylbenz[a]anthracene (DMBA), and dibenzo[a,l]pyrene (DB[a,l]P). 4-FluoroB[j]F did exhibit significant tumor-initiating activity at doses of 10 and 25 nmol per mouse, inducing a 45 and 60% incidence of tumor-bearing mice with an average of 0.75 and 1.65 tumors per mouse, respectively. While B[a]P was not tumorigenic at these doses, DMBA and DB[a,l]P exhibited significant tumorigenic activity at doses of 1, 4, 10, and 25 nmol per mouse. DB[a,l]P induced a 95% incidence of tumor-bearing mice with an average of 5.0 tumors per mouse at a total initiator dose of 1 nmol. DMBA at this dose produced an 85% incidence of tumor-bearing mice with an average of 1.30 tumors per mouse. The results of these initiation promotion bioassays clearly demonstrate that 4-fluoroB[j]F is significantly more active than B[j]F, 10-fluoroB[j]F and B[a]P and less active than either DMBA or DB[a,l]P as a tumor initiator on mouse skin.

32P-Postlabeling analysis of DNA adducts from non-alternant PAH using thin-layer and high performance liquid chromatography

DNA adduct formation in vivo in mouse skin following a single topical application of benzo[a]fluoranthene (BbF), benzo[j]fluoranthene (BjF), benzo[k]fluoranthene (BkF), or indeno[1,2,3-cd]pyrene (IP) was investigated in female CD-1 mice using 32P-postlabeling analysis. Distinct adduct profiles were detected for each of the non-alternant hydrocarbons examined. Two adducts, one major and one minor, were detected using polyethyleneiminecellulose (PEI-cellulose) thin-layer chromatography (TLC) for BbF and BjF while a single major adduct was detected for BkF and IP. The relative extent of binding to mouse skin DNA was in the order BbF greater than BjF greater than BkF greater than IP. 32P-Postlabeled DNA adducts separated by PEI-cellulose TLC were further analyzed by high performance liquid chromatography (HPLC). A single radioactive peak was detected for 32P-labeled DNA adducts of BjF and BkF. Three general areas of radioactivity were detected when 32P-labeled DNA adducts of BbF were separated on HPLC.

Role of activated oxygen species in benzo[a]pyrene: DNA adduct formation in vitro

The role of several activated oxygen species in the oxidation and binding of B[a]P to calf thymus DNA in vitro was investigated. B[a]P was reacted with calf thymus DNA in the presence and absence of scavengers of active oxygen species. Reactions were performed in the dark at 37 degrees C for 30 min in a buffered aqueous solution with 250 micrograms of calf thymus DNA. The levels of B[a]P:DNA adducts formed were determined using the 32P-postlabeling assay. B[a]P:DNA adduct levels ranged from 1.5-2.6 and 0.25 pmol adducts/mg DNA in reactions with 120 or 12 nmol of B[a]P, respectively. The addition of scavengers of reactive oxygen species to reaction mixtures resulted in a considerable decrease in the levels of DNA adducts formed in comparison to control reactions. Reactions performed with 500 units catalase or 100 units superoxide dismutase significantly inhibited DNA adduct formation. In these reactions adduct levels were 32 and 48% of control levels, respectively. The addition of both catalase and superoxide dismutase to reactions inhibited adduct formation by 95% relative to control reactions. A decrease in adduct levels was also observed when reactions were performed with citrate-Fe3+ chelate, a scavenger of superoxide. In reactions with 50 mM mannitol and 50 mM sodium benzoate, both of which are hydroxyl radical scavengers, adduct formation was significantly inhibited with adduct levels being 30 and 51% of control values, respectively. Adduct levels were decreased to 26% of control values in reactions with 10 mM 2,5-dimethylfuran, a scavenger of singlet oxygen.(ABSTRACT TRUNCATED AT 250 WORDS)

Bioassay of quinoline, 5-fluoroquinoline, carbazole, 9-methylcarbazole and 9-ethylcarbazole in newborn mice

Quinoline and carbazole are among the more prevalent aza-arenes present as components of environmental pollutants. Both of these aza-arenes are hepatocarcinogenic to mice when administered in the diet. The hepatocarcinogenic potential of quinoline is consistent with its mutagenic activity in Salmonella typhimurium TA100 and potential to induce unscheduled DNA synthesis (UDS) in rat hepatocytes. Structure-activity studies with fluorinated quinolines indicate that the presence of a fluorine atom at the 5-position of quinoline may inhibit detoxification and result in enhanced genotoxic potency. Quinoline and 5-fluoroquinoline were assayed in newborn CD-1 mice at a total dose of 1.75 mumol to establish their relative tumorigenic activity. Liver tumours developed in 60 and 90% of the male newborn mice treated with quinoline and 5-fluoroquinoline, respectively. The majority of liver tumours observed among the quinoline-treated mice were classified as adenomas. In contrast, liver carcinomas developed in most of the male mice treated with 5-fluoroquinoline. Unlike the well established genotoxic potential of quinoline, there is limited evidence for carbazole having either genotoxic or carcinogenic activity. Whereas carbazole is not mutagenic towards several strains of S. typhimurium, both 9-methylcarbazole and 9-ethylcarbazole are active as mutagens in S. typhimurium TA100. Carbazole, 9-methylcarbazole and 9-ethylcarbazole were assayed in primary rat hepatocytes to assess their relative potential to induce UDS in rat hepatocytes; only 9-ethylcarbazole did so. These carbazole derivatives were also assayed in newborn CD-1 mice at a total dose of 1.75 mumol. Neither carbazole nor either of these 9-alkylcarbazoles produced a significant tumorigenic response in this bioassay system.

Biochemical effects of manufactured gas plant residue following ingestion by B6C3F1 mice

The toxic potential of manufactured gas plant residue (MGP) given in the diet to male and female B6C3F1 mice was evaluated. In addition, the bioavailability of chemical components of MGP were also investigated by monitoring polycyclic aromatic hydrocarbon (PAH) metabolites in urine and DNA adduct formation in forestomach and lung tissue. Basal gel diets containing 0.05, 0.25, 0.50% MGP or 0.005% benzo[a]pyrene (BaP) were fed to animals for 94 and 185 d. Mice readily consumed adulterated diets without any evidence of acute toxicity. The total amount of MGP and BaP consumed by mice ranged from 118 to 2604 mg and from 12 to 29 mg, respectively. Male mice fed a control or BaP diet and female mice fed a 0.05% MGP diet had the highest body weight gains. Male and female mice fed a 0.50% MGP diet had the lowest body weight gains. The bioavailability of chemical components of MGP was evaluated by monitoring the urinary excretion of PAH metabolites by male mice fed a 0.25% MGP diet. 1-Hydroxypyrene was determined by high-performance liquid chromatography analysis to be the major fluorescent metabolite excreted by mice throughout the 185 d of diet administration. At necropsy, no chemical-related gross lesions were detected. In addition, no treatment-related microscopic lesions were evident in tissues obtained from animals fed a 0.50% MGP- or BaP-adulterated diet. The 32P-postlabeling assay was used to evaluate MGP- and BaP-induced DNA adduct formation in lung and forestomach tissue. The level of DNA adducts formed from the chemical components of MGP paralleled the amount of material ingested by animals. Lung DNA adduct levels were considerably higher than forestomach levels when mice ingested a 0.25% or 0.50% MGP diet. These studies demonstrate that the continuous ingestion of MGP or BaP for 185 d does not result in acute toxicity or chemical-related lesions at doses up to 0.50% MGP or 0.005% BaP.

Relative tumor initiating activity of benzo[a]fluoranthene, benzo[b]fluoranthene, naphtho[1,2-b]fluoranthene and naphtho[2,1-a]fluoranthene on mouse skin

The tumor-initiating activities of benzo[a]fluoranthene (BaF), benzo[b]fluoranthene (BbF), naphtho[1,2-b]fluoranthene (NbF) and naphtho[2,1-a]fluoranthene (NaF) were evaluated on the skin of female CD-1 mice. Each of these polycyclic aromatic hydrocarbons was assayed at total initiation doses of 1.0 and 4.0 mumol/mouse. These hydrocarbons were applied in 10 subdoses administered every other day. Promotion commenced 10 days after the last initiator dose and consisted of thrice weekly application of 2.5 micrograms of tetradecanoylphorbol acetate for 20 weeks. BbF was the most potent tumor initiator inducing a 100% incidence of tumor-bearing mice with an average of 8.5 tumors per mouse at a total initiator dose of 1.0 mumol. NaF was slightly more active as a tumor initiator than either NbF or BaF. NaF induced a 90% incidence of tumor-bearing mice with an average of 5.9 tumors per mouse at a total initiator dose of 1.0 mumol. BaF and NbF at a total initiator dose of 4.0 mumol exhibited similar tumor-initiating activity with both inducing a 90% incidence of tumor-bearing mice with an average of 4.3 and 6.6 tumors per mouse, respectively. However, at a total initiator dose of 1.0 mumol, BaF and NbF induced a 95% and 65% incidence of tumor-bearing mice with an average of 3.3 and 2.5 tumors per mouse, respectively.

Carcinogenicity of Coal Tars: A Multidisciplinary Approach

Abstract Coal tars, which are byproducts of coal gasification, are complex mixtures of chemicals including polycyclic aromatic hydrocarbons. Occupational exposure to coal tars is associated with a higher risk of cancer. The risk posed by environmental coal tars, and related oil tars, which may involve prolonged exposures to low doses, is not known. While this risk may be inferred from a knowledge of the concentration and potency of known carcinogenic components, the complexity and incomplete chemical analysis of coal tars lead to estimates with large uncertainties. In order to assess risk with greater certainty, laboratory studies were conducted to evaluate cancer induction following oral administration of coal tars. The appropriateness of these animal data to humans was evaluated in studies of the effects of these tars on genotoxicity, metabolism and gene expression. Our data suggest that the potency of coal tars cannot be predicted accurately from an understanding of the biological or carcinogenic effec...

7H-BENZO[C]FLUORENE: A POTENT SYSTEMIC LUNG CARCINOGEN

7H-Benzo[c]fluorene (B[c]F) is a major lung deoxyribonucleic acid (DNA) adductor in mice treated with coal tar suggesting that B[c]F may be capable of inducing lung tumors. This study evaluated the tumorigenic potential of B[c]F in comparison to benzo[a]pyrene (B[a]P) using the A/J mouse model. Female A/J mice 7 weeks of age were administered B[c]F or B[a]P (100 mg/kg) by i.p. injection. These mice were fed Purina Rodent 2001 diet for the remainder of the study. Groups of mice were also fed diets containing B[c]F (397 or 27 μmol/kg diet) or B[a]P (397 μmol/kg diet). In addition, a mixture of 20 synthetic polycyclic aromatic hydrocarbons (PAHs) known to be present in coal tar was also fed to mice in the presence or absence of B[c]F. A basal gel diet system was used to administer hydrocarbons within the diet. Mice were maintained on control or adulterated diets for 260 days. B[c]F administered i.p. induced multiple lung tumors in 92% of the treated mice, with an average of 4.0 tumors per mouse. Similarly, B[a]P administered i.p. induced an average of 6.7 tumors per mouse in 90% of the treated mice. The highest level of lung tumor induction was observed in mice fed 397 μmol/kg diet B[c]F. A 100% tumor incidence and an average of 46 lung tumors per mouse was observed. In contrast, mice fed a diet containing 397 μmol/kg of B[a]P, had a 77% tumor incidence with an average of 1.4 tumors per mouse. Mice fed a 27 μmol/kg B[c]F diet, or the mixture of synthetic hydrocarbons with or without B[c]F resulted in tumor incidences and multiplicity not different from controls. Forestomach lesions were greatest in mice treated with B[a]P. Evaluation of chemical:DNA adduct formation in mice indicates that B[c]F is better than B[a]P in forming lung DNA adducts when fed to mice at 397 μmol/kg food. These results demonstrate that B[c]F is tumorigenic in lung of mice when administered by i.p. injection and in particular when fed to mice in the diet. These data strongly suggest that B[c]F is a systemic carcinogen that likely contributes to the potent mouse lung tumorigenicity previously demonstrated with coal tar when fed to mice.