Kaarthik John

Polycyclic Aromatic Hydrocarbon (PAH) Exposure and DNA Adduct Semi-Quantitation in Archived Human Tissues

Polycyclic aromatic hydrocarbons (PAHs) are combustion products of organic materials, mixtures of which contain multiple known and probable human carcinogens. PAHs occur in indoor and outdoor air, as well as in char-broiled meats and fish. Human exposure to PAHs occurs by inhalation, ingestion and topical absorption, and subsequently formed metabolites are either rendered hydrophilic and excreted, or bioactivated and bound to cellular macromolecules. The formation of PAH-DNA adducts (DNA binding products), considered a necessary step in PAH-initiated carcinogenesis, has been widely studied in experimental models and has been documented in human tissues. This review describes immunohistochemistry (IHC) studies, which reveal localization of PAH-DNA adducts in human tissues, and semi-quantify PAH-DNA adduct levels using the Automated Cellular Imaging System (ACIS). These studies have shown that PAH-DNA adducts concentrate in: basal and supra-basal epithelium of the esophagus, cervix and vulva; glandular epithelium of the prostate; and cytotrophoblast cells and syncitiotrophoblast knots of the placenta. The IHC photomicrographs reveal the ubiquitous nature of PAH-DNA adduct formation in human tissues as well as PAH-DNA adduct accumulation in specific, vulnerable, cell types. This semi-quantative method for PAH-DNA adduct measurement could potentially see widespread use in molecular epidemiology studies.

Quantification of phase I / II metabolizing enzyme gene expression and polycyclic aromatic hydrocarbon-DNA adduct levels in human prostate

Studies of migrant populations suggest that dietary and/or environmental factors play a crucial role in the etiology of prostatic adenocarcinoma (CaP). The human prostate consists of the peripheral zone (PZ), transition zone (TZ), and central zone (CZ); CaP occurs most often in the PZ.

Exposure to benzo[a]pyrene of Hepatic Cytochrome P450 Reductase Null (HRN) and P450 Reductase Conditional Null (RCN) mice: Detection of benzo[a]pyrene diol epoxide-DNA adducts by immunohistochemistry and 32P-postlabelling.

Benzo[a]pyrene (BaP) is a widespread environmental carcinogen activated by cytochrome P450 (P450) enzymes. In Hepatic P450 Reductase Null (HRN) and Reductase Conditional Null (RCN) mice, P450 oxidoreductase (Por) is deleted specifically in hepatocytes, resulting in the loss of essentially all hepatic P450 function. Treatment of HRN mice with a single i.p. or oral dose of BaP (12.5 or 125mg/kg body weight) resulted in higher DNA adduct levels in liver (up to 10-fold) than in wild-type (WT) mice, indicating that hepatic P450s appear to be more important for BaP detoxification in vivo. Similar results were obtained in RCN mice. We tested whether differences between hepatocytes and non-hepatocytes in P450 activity may underlie the increased liver BaP-DNA binding in HRN mice. Cellular localisation by immunohistochemistry of BaP-DNA adducts showed that HRN mice have ample capacity for formation of BaP-DNA adducts in liver, indicating that the metabolic process does not result in the generation of a reactive species different from that formed in WT mice. However, increased protein expression of cytochrome b(5) in hepatic microsomes of HRN relative to WT mice suggests that cytochrome b(5) may modulate the P450-mediated bioactivation of BaP in HRN mice, partially substituting the function of Por.

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.

Tamoxifen-DNA adduct formation in monkey and human reproductive organs.

The estrogen analog tamoxifen (TAM), used for adjuvant therapy of breast cancer, induces endometrial and uterine tumors in breast cancer patients. Proliferation stimulus of the uterine endometrium is likely involved in tumor induction, but genotoxicity may also play a role. Formation of TAM-DNA adducts in human tissues has been reported but remains controversial. To address this issue, we examined TAM-DNA adducts in uteri from two species of monkeys, Erythrocebus patas (patas) and Macaca fascicularis (macaque), and in human endometrium and myometrium. Monkeys were given 3-4 months of chronic TAM dosing scaled to be equivalent to the daily human dose. In the uteri, livers and brains from the patas (n = 3), and endometrium from the macaques (n = 4), TAM-DNA adducts were measurable by TAM-DNA chemiluminescence immunoassay. Average TAM-DNA adduct values for the patas uteri (23 adducts/10(8) nucleotides) were similar to those found in endometrium of the macaques (19 adducts/10(8) nucleotides). Endometrium of macaques exposed to both TAM and low-dose estradiol (n = 5) averaged 34 adducts/10(8) nucleotides. To examine TAM-DNA persistence in the patas, females (n = 3) were exposed to TAM for 3 months and to no drug for an additional month, resulting in low or non-detectable TAM-DNA in livers and uteri. Human endometrial and myometrial samples from women receiving (n = 8) and not receiving (n = 8) TAM therapy were also evaluated. Women receiving TAM therapy averaged 10.3 TAM-DNA adducts/10(8) nucleotides, whereas unexposed women showed no detectable TAM-DNA. The data indicate that genotoxicity, in addition to estrogen agonist effects, may contribute to TAM-induced human endometrial cancer.

Benzo[a]pyrene (BP) DNA adduct formation in DNA repair–deficient p53 haploinsufficient [Xpa(−/−)p53(+/−)] and wild-type mice fed BP and BP plus chlorophyllin for 28 days

We have evaluated DNA damage (DNA adduct formation) after feeding benzo[a]pyrene (BP) to wild-type (WT) and cancer-susceptible Xpa(-/-)p53(+/-) mice deficient in nucleotide excision repair and haploinsufficient for the tumor suppressor p53. DNA damage was evaluated by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ES-MS/MS), which measures r7,t8,t9-trihydroxy-c-10-(N (2)-deoxyguanosyl)-7,8,9,10-tetrahydrobenzo[a]pyrene (BPdG), and a chemiluminescence immunoassay (CIA), using anti-r7,t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA antiserum, which measures both BPdG and the other stable BP-DNA adducts. When mice were fed 100 ppm BP for 28 days, BP-induced DNA damage measured in esophagus, liver and lung was typically higher in Xpa(-/-)p53(+/-) mice, compared with WT mice. This result is consistent with the previously observed tumor susceptibility of Xpa(-/-)p53(+/-) mice. BPdG, the major DNA adduct associated with tumorigenicity, was the primary DNA adduct formed in esophagus (a target tissue in the mouse), whereas total BP-DNA adducts predominated in higher levels in the liver (a non-target tissue in the mouse). In an attempt to lower BP-induced DNA damage, we fed the WT and Xpa(-/-)p53(+/-) mice 0.3% chlorophyllin (CHL) in the BP-containing diet for 28 days. The addition of CHL resulted in an increase of BP-DNA adducts in esophagus, liver and lung of WT mice, a lowering of BPdG in esophagi of WT mice and livers of Xpa(-/-)p53(+/-) mice and an increase of BPdG in livers of WT mice. Therefore, the addition of CHL to a BP-containing diet showed a lack of consistent chemoprotective effect, indicating that oral CHL administration may not reduce PAH-DNA adduct levels consistently in human organs.

Role of nucleotide excision repair and p53 in zidovudine (AZT)‐induced centrosomal deregulation

The nucleoside reverse transcriptase inhibitor zidovudine (AZT) induces genotoxic damage that includes centrosomal amplification (CA > 2 centrosomes/cell) and micronucleus (MN) formation. Here we explored these end points in mice deficient in DNA repair and tumor suppressor function to evaluate their effect on AZT‐induced DNA damage. We used mesenchymal‐derived fibroblasts cultured from C57BL/6J mice that were null and wild type (WT) for Xpa, and WT, haploinsufficient and null for p53 (6 different genotypes). Dose‐responses for CA formation, in cells exposed to 0, 10, and 100 μM AZT for 24 hr, were observed in all genotypes except the Xpa(+/+)p53(+/−) cells, which had very low levels of CA, and the Xpa(−/−)p53(−/−) cells, which had very high levels of CA. For CA there was a significant three‐way interaction between Xpa, p53, and AZT concentration, and Xpa(−/−) cells had significantly higher levels of CA than Xpa(+/+) cells, only for p53(+/−) cells. In contrast, the MN and MN + chromosomes (MN + C) data showed a lack of AZT dose response. The Xpa(−/−) cells, with p53(+/+) or (+/−) genotypes, had levels of MN and MN + C higher than the corresponding Xpa(+/+) cells. The data show that CA is a major event induced by exposure to AZT in these cells, and that there is a complicated relationship between AZT and CA formation with respect to gene dosage of Xpa and p53. The loss of both genes resulted in high levels of damage, and p53 haploinsufficicency strongly protected Xpa(+/+) cells from AZT‐induced CA damage. Environ. Mol. Mutagen. 55:719–726, 2014. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Abstract 3458: Benzo[a]pyrene (BP)-induced DNA damage is elevated in mice lacking Xeroderma pigmentosum complementation group A (Xpa) and heterozygous for the tumor suppressor gene p53

In developing countries, esophageal cancer is the third most common cancer in men. We have employed a mouse model of esophageal cancer using DNA repair deficient mice. Unlike their wild type counterparts, C57BL/6J mice lacking Xpa and heterozygous for p53 [Xpa(−/−) p53(+/−)] have previously been shown to develop forestomach and esophageal tumors when fed BP (Hoogervorst et al., Carcinogenesis, 2003). Two methodologies were employed to examine BP-induced steady-state levels of DNA damage in esophagi of male mice fed 0 or 100 ppm BP for 4 weeks. HPLC, in conjunction with tandem mass spectrometry (HPLC-MS/MS), was used to measure 10-(deoxyguanosin-N 2 -yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPdG) and a chemiluminiscence immunoassay (CIA), using antiserum elicited against r7,t8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-modified DNA was used to evaluate BP-DNA adducts. In the esophagi of Xpa(−/−) p53(+/−) mice, there were 38.7 ± 4.8 BPdG adducts/10 8 nucleotides (mean ± SE, n=5), as measured by HPLC-MS/MS, while in the wild type mice there were 28.0 ± 1.8 BPdG adducts/10 8 nucleotides (mean ± SE, n=5). Furthermore, complete loss of p53 resulted in the greatest accumulation of BPdG adducts, with 58.1 ± 8.6 BPdG adducts/10 8 nucleotides (n=5) in Xpa(−/−) p53(−/−) mice (p 8 nucleotides (n=3) in the Xpa(−/−) p53(+/−) mice, compared to 14.6 ± 3.0 adducts/10 8 nucleotides (n=3) in the wild type mice. Since similar adduct levels were determined using both HPLC-MS/MS and CIA, this indicates that BPdG is the major adduct resulting from BP in mouse esophagus. The higher levels of DNA damage in DNA repair-deficient p53-deficient mice, compared to their wild type counterparts, may contribute to the enhanced susceptibility of these mice to esophageal tumor induction by BP. Currently we are examining the role of chlorophyllin, a chemopreventive agent, in reducing BP-induced DNA damage levels and tumor formation in this model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3458.