Kathy LaDow

Biotransformation of benzo[a]pyrene and other polycyclic aromatic hydrocarbons and heterocyclic analogs by several green algae and other algal species under gold and white light.

This laboratory has shown that the metabolism of benzo[a]pyrene (BaP), a carcinogenic polycyclic aromatic hydrocarbon (PAH), by a freshwater green alga, Selenastrum capricornutum, under gold light proceeds through a dioxygenase pathway with subsequent conjugation and excretion. This study was undertaken to determine: (1) the effects of different light sources on the enzymatic or photochemical processes involved in the biotransformation of BaP over a dose range of 5-1200 mg/l; (2) the phototoxicity of carcinogenic PAHs and mutagenic quinones to a green alga; (3) the ability of other algal systems to metabolize BaP. Cultures were exposed to different doses of BaP for 2 days at 23 degrees C under gold, white or UV-A fluorescent light on a diurnal cycle of 16 h light, 8 h dark. Under gold light, metabolites of BaP produced by Selenastrum capricornutum were the dihydrodiols of which the 11,12-dihydrodiol was the major metabolite. Under white light, at low doses, the major metabolite was the 9,10-dihydrodiol. With increasing dose, the ratio of dihydrodiols to quinones decreased to less than two. With increasing light energy output, from gold to white to UV-A in the PAH absorbing region, BaP quinone production increased. Of other carcinogenic PAHs studied, only 7H-dibenz[c,g]carbazole was as phototoxic as BaP while 7,12-dimethylbenz[a]anthracene, dibenz[a,j]acridine and non-carcinogenic PAHs, anthracene and pyrene, were not phototoxic. The 3,6-quinone of BaP was found to be highly phototoxic while quinones that included menadione, danthron, phenanthrene-quinone and hydroquinone were not. The data suggest that the phototoxicity of BaP is due to photochemical production of quinones; the 3,6-quinone of BaP is phototoxic and is probably the result of the production of short lived cyclic reactive intermediates by the interaction of light with the quinone. Lastly, only the green algae, Selenastrum capricornutum, Scenedesmus acutus and Ankistrodesmus braunii almost completely metabolized BaP to dihydrodiols. The green alga Chlamydomonas reinhardtii, the yellow alga Ochromonas malhamensis, the blue green algae Anabaena flosaquae and euglenoid Euglena gracilis did not metabolize BaP to any extent. The data indicate that algae are important in their ability to degrade PAHs but the degradation is dependent on the dose of light energy emitted and absorbed, the dose of PAHs to which the algae are exposed, the phototoxicity of PAHs and their metabolite(s) and the species and strain of algae involved. All of these factors will be important in assessing the degradation and detoxification pathways of recalcitrant PAHs by algae.

Inhibition of VEGFR2 prevents DMBA-induced mammary tumor formation

Preinvasive mammary pathologies in humans and rat chemical carcinogenesis model systems have an increased microvascular density relative to normal tissue. This suggests the possibility of preventing invasive breast cancer by inhibiting angiogenesis. Vascular endothelial cell growth factor (VEGF) is a potent angiogenic growth factor, commonly involved in tumor-induced angiogenesis. Here, we show that both VEGF and VEGFR2 expression increase with histological progression to invasive disease in the rat 7,12-dimethylbenz[a]anthracene (DMBA) model. Other VEGF receptors, VEGFR1, neuropilin 1 and neuropilin 2, are constitutively expressed throughout progression. To examine whether VEGF signaling is functionally relevant to tumor-induced endothelial tubule formation in vitro and for tumor formation in vivo, we utilized the VEGFR2 inhibitor, ZD6474. In vitro endothelial cell tubulogenesis induced by isolated mammary organoids or carcinoma in situ from DMBA-treated rats is inhibited by ZD6474, in a dose-dependent fashion. The administration of ZD6474 to DMBA-treated rats inhibits the formation of atypical ductal hyperplasia and carcinoma in situ by greater than 95% (P<0.05), when administered 1 week or 6 weeks post-DMBA initiation. Invasive disease was absent in all ZD6474 cohorts. These data support the hypothesis that progression of DMBA-induced preinvasive mammary pathologies to palpable disease requires angiogenesis via a VEGF-dependent mechanism.

DMBA-induced mammary pathologies are angiogenic in vivo and in vitro.

We have previously shown that human pre-invasive diseases of the breast are angiogenic. In addition, normal epithelium from women with coincident or subsequent invasive breast cancer is more vascular than normal epithelium from women with no breast cancer. To develop a model in which to study the regulation of angiogenesis in pre-invasive mammary pathologies, we examined 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tissues for the presence of neovascularization in pre-invasive histopathologies. These studies included morphometric analysis of tissue vascularity in pre-invasive lesions. In addition, we isolated fresh tumors and histologically normal epithelium (organoids) from DMBA or vehicle-treated control rats to test their ability to induce endothelial cell tubule formation in vitro. Finally, we examined tumors for their ability to produce vascular endothelial cell growth factor. The morphometric studies documented that with epithelial progression, the ability of individual cells to elicit angiogenesis increases. The in vitro studies showed that isolated tumors from these animals stimulate angiogenesis. Furthermore, normal epithelium from DMBA-treated rats is more angiogenic than epithelium from control animals. Finally, DMBA-induced tumors produce vascular endothelial growth factor (VEGF) mRNA, therefore, DMBA-induced mammary tumorigenesis is one model in which to test the dependency of progression on angiogenesis.

Expression of Angiogenic Factors Is Upregulated in DMBA-Induced Rat Mammary Pathologies

Objective: In the 7,12-dimethylbenz[a]anthracene (DMBA) model of rat mammary carcinogenesis, microvascular density and angiogenic potential increase with progression from normal to invasive disease, but the mechanisms involved are unknown. Using RT-PCR, we determined the expression of angiogenic regulators in DMBA-induced intraductal hyperplasia (IDP), carcinoma in situ (CIS), invasive tumors (INV), as well as normal tissue. Methods: RT-PCR was performed on frozen tissue sections of each type of pathology for factors known to regulate angiogenesis in other systems. Results: MMP-2, MMP-9, uPA, PAI-1, IGF-2, BFGF, VEGF, ANG-1, IRS-1, and TSP-1 were significantly (p ≤ 0.05) upregulated in CIS and INV, whereas TIMP-1, ANG-2, MASPIN, IGF1-R and HBEGF were unchanged. IGF-1 was uniquely elevated in IDP. SPARC was downregulated in CIS. Inhibition of IGF-1R by the tyrphostin, AG1024, blocked endothelial tubulogenesis in vitro, confirming that IGF-1 functions as a regulator of angiogenesis. Conclusions: These data support the involvement of specific angiogenic mediators in mammary tumor formation. Angiogenesis at different stages of tumorigenesis may be regulated by unique factors.

TNP-470 inhibits 7,12-dimethylbenz[a]anthracene-induced mammary tumor formation when administered before the formation of carcinoma in situ but is not additive with tamoxifen.

In many women pathologic lesions, such as hyperplasia and carcinoma in situ, precede invasive breast cancer. We have shown that tissue vascularity increases with histologic progression to invasive disease. Similarly, in the well-characterized 7,12-dimethylbenz[a]anthracene (DMBA) model of mammary tumorigenesis, preinvasive lesions exhibit increased vascularity with progression. Using this model we asked whether inhibition of angiogenesis would block progression and if so, at which stage. We treated rats with DMBA followed by the potent angiogenic inhibitor, TNP-470, and/or tamoxifen starting 1 day or 6 weeks later. Histopathology and in vitro angiogenic potential of mammary organoids were evaluated 3 months after DMBA. All statistical tests were two-sided. Early TNP-470 and tamoxifen treatment inhibited the formation of carcinoma in situ (p < 0.001) and invasive disease (p < 0.001). However, their effects were not additive, despite their unique mechanisms of action. TNP-470 administration begun at the time of microscopic carcinoma in situ formation was unable to prevent the further development of carcinoma in situ or invasive breast cancer, whereas tamoxifen was highly effective (p = 0.001). There was no added benefit of combining TNP-470 and tamoxifen. TNP-470 therapy, unlike tamoxifen, did not inhibit the angiogenic potential of DMBA-treated normal mammary organoids, supporting its lack of a direct effect on the epithelium. These data provide proof-in-principle that inhibition of angiogenesis early in mammary tumorigenesis prevents mammary tumor formation in a hormone-sensitive model, indicating that angiogenesis is a potential target for cancer chemoprevention. Interactions with other chemopreventive strategies and the timing of administration must be thoroughly examined in vivo.

Reduction of a 7,12-dimethylbenz[a]anthracene DNA adduct in rat mammary tissue in vivo when pretreated with tamoxifen.

Tamoxifen (TAM), an antiestrogenic compound, has been approved for the treatment of breast cancer in high risk women. TAM has been shown to be an effective agent for prevention of breast cancer in women of varying degrees of risk and has been proposed to be used prophylactically in women whose genetic background suggests a high risk for breast cancer. However, it is not known whether TAM given prophylactically will alter the response of women to carcinogens from common environmental exposures such as tobacco smoke. Therefore, we studied the effects of TAM pretreatment on mammary DNA adducts of the model carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA), in the female rats to assess whether TAM would alter the adduct pattern of DMBA. TAM (0.3 mg/day) was given for 7 days prior to a single 20 mg DMBA gavage treatment that is considered a carcinogenic dose in the rat. At 7 days post-DMBA, there was a reduction in the major DMBA-DNA adduct and a significant reduction in the minor DMBA-DNA adduct in mammary glands (P=0.002) of TAM pretreated rats compared to control rats. These data indicate that TAM may alter either metabolic steps in the formation of DNA binding species and/or enhance adduct removal. These data suggest that TAM given to women prior to the development of breast cancer may modulate the impact of environmental exposures, for example, tobacco smoke. Furthermore, research is needed to determine if modulation will be positive, or negative as in the current study.

GENE EXPRESSION PROFILING OF BLOOD TO PREDICT THE ONSET OF LEUKEMIA

No studies have tested the hypothesis that the onset of a disease can be predicted by gene expression profiling. The AKR/J mouse strain, which spontaneously develops acute T cell lymphatic leukemia, was used to implement a novel strategy to generate global gene expression profiles of WBCs at different time points. The experimental approach was bias free because it was unknown as to which individuals in the mouse population would eventually develop the disease. Our results suggest that profiling WBC gene expression may be an effective means for the very early diagnosis of disease in humans.

PRELIMINARY FINDINGS THAT KEROSENE ALTERS THE DISTRIBUTION OF TOPICALLY APPLIED BENZO[A]PYRENE IN MICE

The dermal route is important in occupational exposure to polycyclic aromatic compounds (PACs), but other organs may be affected. We reported that kerosene-cleaning following treatment with used engine oil increased DNA adducts in the lungs of mice viz. animals treated with used oil alone. To determine the mechanism we topically applied 3 H-BAP(100 nmol in 25 μL acetone) and washed half the mice with 25 μL kerosene 1 h after carcinogen application. Groups of four mice were sacrificed from 1 to 72 h after treatment. Lung, liver, and skin were harvested. The fraction of the radiolabel remaining in the skin of animals treated with benzo[a]pyrene (BAP) and washed with kerosene was significantly less than those not washed, beginning at 24 h (p < .05). Fractional distribution to the lungs and livers of these animals became significantly elevated. Kerosene increased transdermal water loss. Kerosene treatment compromises dermal barrier function, enhances carcinogen absorption, and alters organ distribution.

Effects of DMBA Preparation on DMBA-Induced Rat Mammary Carcinogenesis

We compared two protocols for 7,12-dimethylbenz[ a ]anthracene (DMBA)-induced mammary carcinogenesis. DMBA (20 mg/ml) was prepared at room temperature or at 60°C and administered by gavage to 50-day-old Sprague-Dawley rats. Two days post-DMBA, mammary epithelial DNA was 32 P-postlabeled. At 12 weeks post-DMBA, tumor DNA was screened for H- ras codon 61 mutations using an enriched polymerase chain reaction (EPCR). The number of in situ carcinomas (3.0 - 2.7 vs. 2.5 - 2.7), invasive carcinomas (1.1 - 1.6 vs. 0.6 - 0.8), and H- ras codon 61 mutations (34 - 13% vs. 23 - 13%) were statistically not different ( p = 0.05) between the groups. The relative adduct levels in mammary organoids were significantly ( p h 0.001) greater in the 60°C (568 - 158) than the room temperature (150 - 130) group. While DMBA-DNA adduct levels differed significantly between these protocols, this did not correlate with significant differences in tumor pathologies or H- ras codon 61 mutations.

Comparison of N-Heterocyclic Aromatics Dibenzo[c,g]Carbazole and Dibenz[a,j]Acridine : Metabolism, DNA Binding and Mutation

Abstract N-Heterocyclic aromatics, such as carbazole and acridine derivatives, are environmental carcinogenic pollutants. Examples of these compounds are 7H-dibenzo[c,g]carbazole (DBC) and dibenz[a,j]acridine (DBA). The ionization potential (IP) for DBC is lower than for DBA. DBC is metabolized in lung and liver by way of phenols or directly through radical cations. DBC-induced liver and lung tumors have mutations in the 61st codon of ras. DBA is metabolized in skin by way of a diol-epoxide of DBA. DBA-induced skin tumors have mutations in 12th, 13th and 61st codons of ras. In summary, the metabolic activation of DBC proceeds through different adduction pattern pathways than does DBA and leads to different ras mutational spectra.