Acute treatment with kerosene damages the dermal barrier and alters the distribution of topically applied benzo(a)pyrene in mice.

The dermal route is important in many occupational exposures. Some materials may reduce the barrier function of the skin to enhance absorption and effect on internal organs. We have reported previously that kerosene cleaning following treatment with used engine oil increased DNA adduct levels in the lungs of mice compared with animals treated with used oil alone. To investigate what other physiological parameters might be affected by kerosene, we conducted in vitro and in vivo measurements of skin barrier function. We also topically applied (3)H-BAP(100 nM in 25 µL acetone) and washed half the mice with 25 µL kerosene 1 hr after carcinogen application. Groups of four mice were euthanized from 1 to 72 hr after treatment. Skin, lungs, and livers were harvested from each animal and stored separately. Kerosene application reduced the barrier function of the skin in vitro beyond the effect of the acetone vehicle and the vehicle plus BAP. In vivo studies indicated that kerosene treatment reduced the barrier function at 4 and 8 hr post application and that the barrier function recovered at 24 hr after a single treatment. The fraction of the radiolabel remaining in the skin of animals treated with (3)H-BAP and washed with kerosene was significantly less than those not washed, beginning at 24 hr (p< 0.05). Fractional distribution to the lungs and livers of these animals became significantly elevated at this time. Kerosene treatment compromises dermal barrier function and the ability of the skin to retain water, enhances carcinogen absorption, and alters organ distribution. This appears to contribute to the increase in BAP DNA adducts we reported earlier.

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.

Enhanced degradation of benzo[a]pyrene by Mycobacterium sp. in conjunction with green alga.

Previous work in this laboratory has confirmed that the bacteria Mycobacterium sp. strain RJGII.135 and Sphingomonas yanoikuyae strain B1 and the green alga Selanastrum capricornutum strain UTEX 1648 degrade benzo[a]pyrene (BaP) to various BaP intermediates. S. capricornutum was first grown with BaP for 4 days. The organic extract of this media was then introduced into separate cultures of strain RJGII.135 and strain B1; separate cultures were grown with BaP for comparison. Cultures grown with BaP and those grown with the algal/BaP extract showed similar mineralization patterns. The quantity of total metabolites formed was greater in bacterial cultures grown with the algal/BaP extract than those grown with BaP alone. For strain RJGII.135, only 27% of the original BaP remained in cultures grown with the algal/BaP extract; 59% remained in cultures grown with BaP. For strain B1, only 6% of the original BaP remained in cultures grown with the algal/BaP extract; 38% remained in cultures grown with BaP. These results indicate that strategies utilizing organisms together may be necessary in being able to degrade large, recalcitrant polycyclic aromatic hydrocarbons (PAHs) such as BaP.

Impact of Cyp1a2 or Ahr gene knockout in mice: implications for biomonitoring studies.

UNLABELLED: Studies of the impact of phase 1 enzyme polymorphisms on genetic damage have yielded mixed results. We studied how genetic damage would be altered when specific genes were ablated under low dose conditions. METHODS: Knockouts (KO) were generated from c57bl6/J mice with mutations in Cyp1a2 or Ahr receptor that eliminated gene product function. Animals were treated topically with either 4-aminobiphenyl (4ABP) 10mg/kg, benzo(a)pyrene (BaP) 33.3mg/kg or dibenzo(c,g)carbazole (DBC) 8 mg/kg, and sacrificed after 24h. DNA from livers, skin and/or urinary bladders were isolated and (32)P-post labelled. RESULTS: Cyp1a2-/- mice did not differ in 4ABP DNA adduct levels in either urinary bladder or liver compared to wildtype. There was a sex difference in the organ affected. Cyp1a2 knockout reduced skin BAP adduct levels 50% and AHR knockout reduced skin BAP adduct levels by 90%. There was no impact of either knockout on the levels of DBC-DNA adducts in any tissue. CONCLUSIONS: Ablation of specific metabolizing enzymes had compound- and tissue-specific effects in mice. Phenotypic variability in single CYP enzymes may have minor impact in humans at low doses, but variation in the ability to induce the family of CYPs may have a greater impact.

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 < or =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.

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.

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.