Linda Adams

CD34 Expression by Hair Follicle Stem Cells Is Required for Skin Tumor Development in Mice

The cell surface marker CD34 marks mouse hair follicle bulge cells, which have attributes of stem cells, including quiescence and multipotency. Using a CD34 knockout (KO) mouse, we tested the hypothesis that CD34 may participate in tumor development in mice because hair follicle stem cells are thought to be a major target of carcinogens in the two-stage model of mouse skin carcinogenesis. Following initiation with 200 nmol 7,12-dimethylbenz(a)anthracene (DMBA), mice were promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 20 weeks. Under these conditions, CD34KO mice failed to develop papillomas. Increasing the initiating dose of DMBA to 400 nmol resulted in tumor development in the CD34KO mice, albeit with an increased latency and lower tumor yield compared with the wild-type (WT) strain. DNA adduct analysis of keratinocytes from DMBA-initiated CD34KO mice revealed that DMBA was metabolically activated into carcinogenic diol epoxides at both 200 and 400 nmol. Chronic exposure to TPA revealed that CD34KO skin developed and sustained epidermal hyperplasia. However, CD34KO hair follicles typically remained in telogen rather than transitioning into anagen growth, confirmed by retention of bromodeoxyuridine-labeled bulge stem cells within the hair follicle. Unique localization of the hair follicle progenitor cell marker MTS24 was found in interfollicular basal cells in TPA-treated WT mice, whereas staining remained restricted to the hair follicles of CD34KO mice, suggesting that progenitor cells migrate into epidermis differently between strains. These data show that CD34 is required for TPA-induced hair follicle stem cell activation and tumor formation in mice.

Lack of contribution of covalent benzo[a]pyrene-7,8-quinone–DNA adducts in benzo[a]pyrene-induced mouse lung tumorigenesis☆

Benzo[a]pyrene (B[a]P) is a potent human and rodent lung carcinogen. This activity has been ascribed in part to the formation of anti-trans-7,8-dihydroxy-7,8-dihydroB[a]P-9,10-epoxide (BPDE)-DNA adducts. Other carcinogenic mechanisms have been proposed: (1) the induction of apurinic sites from radical cation processes, and (2) the metabolic formation of B[a]P-7,8-quinone (BPQ) that can form covalent DNA adducts or reactive oxygen species which can damage DNA. The studies presented here sought to examine the role of stable BPQ-DNA adducts in B[a]P-induced mouse lung tumorigenesis. Male strain A/J mice were injected intraperitoneally once with BPQ or trans-7,8-dihydroxy-7,8-dihydroB[a]P (BP-7,8-diol) at 30, 10, 3, or 0mg/kg. Lungs and livers were harvested after 24h, the DNA extracted and subjected to (32)P-postlabeling analysis. Additional groups of mice were dosed once with BPQ or BP-7,8-diol each at 30 mg/kg and tissues harvested 48 and 72 h later, or with B[a]P (50mg/kg, a tumorigenic dose) and tissues harvested 72 h later. No BPQ or any other DNA adducts were observed in lung or liver tissues 24, 48, or 72 h after the treatment with 30 mg/kg BPQ. BP-7,8-diol gave BPDE-DNA adducts at all time points in both tissues and B[a]P treatment gave BPDE-DNA adducts in the lung. In each case, no BPQ-DNA adducts were detected. Mouse body weights significantly decreased over time after BPQ or BP-7,8-diol treatments suggesting that systemic toxicity was induced by both agents. Model studies with BPQ and N-acetylcysteine suggested that BPQ is rapidly inactivated by sulfhydryl-containing compounds and not available for DNA adduction. We conclude that under these treatment conditions BPQ does not form stable covalent DNA adducts in the lungs or livers of strain A/J mice, suggesting that stable BPQ-covalent adducts are not a part of the complex of mechanisms involved in B[a]P-induced mouse lung tumorigenesis.

A quantitative comparison of dibenzo[a,l]pyrene-DNA adduct formation by recombinant human cytochrome P450 microsomes†

Dibenzo[a,l]pyrene (DB[a,l]P), an extremely potent environmental carcinogen, is metabolically activated in mammalian cells and microsomes through the fjord‐region dihydrodiol, trans‐DB[a,l]P‐11,12‐diol, to syn‐ and anti‐DB[a,l]P‐11,12‐diol‐13,14‐epoxides (syn‐ and anti‐DB[a,l]PDEs). The role of seven individual recombinant human cytochrome P450s (1A1, 1A2, 1B1, 2B6, 2C9, 2E1, and 3A4) in the metabolic activation of DB[a,l]P and formation of DNA adducts was examined by using 32P postlabeling, thin‐layer chromatography, and high‐pressure liquid chromatography. We found that, in the presence of epoxide hydrolase, only P450 1A1 and P450 1B1 catalyzed the formation of DB[a,l]PDE‐DNA adducts and several unidentified polar adducts. Human P450 1A1 catalyzed the formation of DB[a,l]PDE‐DNA adducts and unidentified polar adducts at rates threefold and 17‐fold greater than did human P450 1B1 (256 fmol/h/nmol P450 versus 90 fmol/h/nmol P450 and 132 fmol/h/nmol P450 versus 8 fmol/h/nmol P450, respectively). P450 1A1 DNA adducts were derived from both anti‐ and syn‐DB[a,l]PDE at rates of 73 fmol/h/nmol P450 and 51 fmol/h/nmol P450, respectively. P450 1B1 produced adducts derived from anti‐DB[a,l]PDE at a rate of 82 fmol/h/nmol, whereas only a small number of adducts were derived from syn‐DB[a,l]PDE (0.4 fmol/h/nmol). These results demonstrated the potential of human P450 1A1 and P450 1B1 to contribute to the metabolic activation and carcinogenicity of DB[a,l]P and provided additional evidence that human P450 1A1 and 1B1 differ in their stereospecific activation of DB[a,l]P. Mol. Carcinog. 26:74–82, 1999. Published 1999 Wiley‐Liss, Inc.

Assessment of multiple types of DNA damage in human placentas from smoking and nonsmoking women in the Czech Republic.

Three classes of DNA damage were assessed in human placentas collected (2000–2004) from 51 women living in the Teplice region of the Czech Republic, a mining area considered to have some of the worst environmental pollution in Europe in the 1980s. Polycyclic aromatic hydrocarbon (PAH)‐DNA adducts were localized and semiquantified using immunohistochemistry (IHC) and the Automated Cellular Imaging System (ACIS). More generalized DNA damage was measured both by 32P‐postlabeling and by abasic (AB) site analysis. Placenta stained with antiserum elicited against DNA modified with 7β,8α‐dihydroxy‐9α,10α‐epoxy‐7,8,9,10‐tetrahydro‐benzo[a]pyrene (BPDE) revealed PAH‐DNA adduct localization in nuclei of the cytotrophoblast (CT) cells and syncytiotrophoblast (ST) knots lining the chorionic villi. The highest levels of DNA damage, 49‐312 PAH‐DNA adducts/108 nucleotides, were found by IHC/ACIS in 14 immediately fixed placenta samples. An additional 37 placenta samples were stored frozen before fixation and embedding, and because PAH‐DNA adducts were largely undetectable in these samples, freezing was implicated in the loss of IHC signal. The same placentas (n = 37) contained 1.7–8.6 stable/bulky DNA adducts/108 nucleotides and 0.6–47.2 AB sites/105 nucleotides. For all methods, there was no correlation among types of DNA damage and no difference in extent of DNA damage between smokers and nonsmokers. Therefore, the data show that DNA from placentas obtained in Teplice contained multiple types of DNA damage, which likely arose from various environmental exposures. In addition, PAH‐DNA adducts were present at high concentrations in the CT cells and ST knots of the chorionic villi. Environ. Mol. Mutagen. 52:58–68, 2011.

Integration of Life-Stage Physiologically Based Pharmacokinetic Models with Adverse Outcome Pathways and Environmental Exposure Models to Screen for Environmental Hazards.

A computational framework was developed to assist in screening and prioritizing chemicals based on their dosimetry, toxicity, and potential exposures. The overall strategy started with contextualizing chemical activity observed in high-throughput toxicity screening (HTS) by mapping these assays to biological events described in Adverse Outcome Pathways (AOPs). Next, in vitro to in vivo (IVIVE) extrapolation was used to convert an in vitro dose to an external exposure level, which was compared with potential exposure levels to derive an AOP-based margins of exposure (MOE). In this study, the framework was applied to estimate MOEs for chemicals that can potentially cause developmental toxicity following a putative AOP for fetal vasculogenesis/angiogenesis. A physiologically based pharmacokinetic (PBPK) model was developed to describe chemical disposition during pregnancy, fetal, neonatal, and infant to adulthood stages. Using this life-stage PBPK model, maternal exposures were estimated that would yield fetal blood levels equivalent to the chemical concentration that altered in vitro activity of selected HTS assays related to the most sensitive vasculogenesis/angiogenesis putative AOP. The resulting maternal exposure estimates were then compared with potential exposure levels using literature data or exposure models to derive AOP-based MOEs.

Association between mutation spectra and stable and unstable DNA adduct profiles in Salmonella for benzo[a]pyrene and dibenzo[a,l]pyrene.

Benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) are two polycyclic aromatic hydrocarbons (PAHs) that exhibit distinctly different mutagenicity and carcinogenicity profiles. Although some studies show that these PAHs produce unstable DNA adducts, conflicting data and arguments have been presented regarding the relative roles of these unstable adducts versus stable adducts, as well as oxidative damage, in the mutagenesis and tumor-mutation spectra of these PAHs. However, no study has determined the mutation spectra along with the stable and unstable DNA adducts in the same system with both PAHs. Thus, we determined the mutagenic potencies and mutation spectra of BP and DBP in strains TA98, TA100 and TA104 of Salmonella, and we also measured the levels of abasic sites (aldehydic-site assay) and characterized the stable DNA adducts ((32)P-postlabeling/HPLC) induced by these PAHs in TA104. Our results for the mutation spectra and site specificity of stable adducts were consistent with those from other systems, showing that DBP was more mutagenic than BP in TA98 and TA100. The mutation spectra of DBP and BP were significantly different in TA98 and TA104, with 24% of the mutations induced by BP in TA98 being complex frameshifts, whereas DBP produced hardly any of these mutations. In TA104, BP produced primarily GC to TA transversions, whereas DBP produced primarily AT to TA transversions. The majority (96%) of stable adducts induced by BP were at guanine, whereas the majority (80%) induced by DBP were at adenine. Although BP induced abasic sites, DBP did not. Most importantly, the proportion of mutations induced by DBP at adenine and guanine paralleled the proportion of stable DNA adducts induced by DBP at adenine and guanine; however, this was not the case for BP. Our results leave open a possible role for unstable DNA adducts in the mutational specificity of BP but not for DBP.

Biotransformation and DNA Adduct Formation of Trans-8,9-Dihydroxy-8,9-Dihydrodibenzo[a, l]Pyrene by Induced Rat Liver and Human CYP1A1 Microsomes

Abstract In order to explain the adduct patterns observed from the human CYP1A1-mediated binding of dibenzo[a, l]pyrene (DB[a, l]P) to DNA, we have investigated the further metabolism and DNA adduct activity of trans-DB[a, l]P-8,9-diol by induced rat liver and human CYP1A1 microsomes. trans-DB[a, l]P-8,9-diol was synthesized and metabolic studies with β-naphthoflavone-induced rat liver microsomes indicated three major metabolites: 2 diastereomers of trans,trans-8,9,11,12-tetrahydro-8,9,11,12-tetrahydroxy-DB[a, l]P and 8,9,13,14-tetrahydro-8,9,13,14-tetrahydroxy-DB[a, l]P. DB[a, l]P when activated by CYP1A1/epoxide hydrase (EH) and calf thymus DNA gave a complex pattern of DNA adducts most of which cochromatograph with syn- and anti-DB[a, l]P fjord region diol epoxide-DNA standards. Two highly polar eluting adducts were also observed, one which cochromatographs with the single major DNA adduct obtained from the CYP1A1/EH activation of trans-DB[a, l]P-8,9-diol. The relative retention time of this adduct suggests either a bis-diol epoxide adduct or a more polar diol epoxide adduct.