• Effects of Smoking Cessation on Eight Urinary Tobacco Carcinogen and Toxicant Biomarkers
  
  Abstract summary: Tobacco carcinogen and toxicant biomarkers, quantitative measurements of exposure and fate of specific tobacco carcinogens and toxicants, could in principle become an important part of a risk algorithm to identify smokers susceptible to lung cancer. They are also important for the evaluation and potentially regulation of new and existing tobacco products.
  
  Authors: Steven G. Carmella, Menglan Chen, Shaomei Han, Joni Jensen, Anna Briggs, Dorothy K. Hatsukami, and Stephen S. Hecht
  
  Laboratory location: Masonic Cancer Center, University of Minnesota, USA
  
  Date: 2009 American Association for Cancer Research (AACR) meeting
  
  
• Associations between cigarette smoking and mitochondrial DNA abnormalities in buccal cells
  
  Abstract summary: DNA alterations in mitochondria are believed to play a role in carcinogenesis, and are found in smoking-related cancers.
  
  Authors: Duanjun Tan, David S Goerlitz, Ramona G. Dumitrescu, Françoise Seillier-Moiseiwitsch, Stephanie M Spernak, Roy Anthony Orden, Jinguo Chen, Radoslav Goldman and Peter G. Shields*
  
  Laboratory location: Cancer Genetics and Epidemiology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA. 20057
  
  Date: 2007 American Association for Cancer Research (AACR) Meeting
  
  
• Toxicological analysis of low-nicotine and nicotine-free cigarettes
  
  Abstract summary: Low-nicotine and nicotine-free cigarettes are commercially available under the brand-name Quest®. Some consumers may believe that these are safer cigarettes, and they may smoke more cigarettes per day or inhale more smoke per cigarette to compensate for low nicotine yields.
  
  Authors: Jinguo Chen,* Higby Richard, † Defa Tian,* Duanjun Tan, * Michael Johnson,* Yingxian Xiao, * Kenneth J Kellar, * Shibao Feng, * Peter G. Shields *
  
  Laboratory location: Cancer Genetics and Epidemiology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA. 20057
  
  Date: 2006 American Association for Cancer Research (AACR) Meeting
  
  
• Somatic mitochondrial DNA mutations and content change in buccal cells of smokers
  
  Abstract summary: mtDNA is particularly susceptible to damage by mutagens. It exhibits 17 times higher rates of mutation than does the nuclear genome and DNA damage persists longer in the mitochondrial genome. The hypothesis in this abstract is that mtDNA damage would be a good biomarker of tobacco smoke exposure.
  
  Authors: DuanJun Tan, Jinguo Chen, David S Goerlitz, Ramona Dumitreascu, Roy Anthony Orden, Radoslav Goldman and Peter G Shields
  
  Laboratory location: Division of Cancer Genetics and Molecular Epidemiology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, 20057
  
  Date: 2008 American Society of Preventive Oncology (ASPO) Annual Meeting; Publication in Cancer Epidemiology Biomarkers and Prevention (CEBP) journal, February 2008
  
  
• Quest nicotine-free cigarette smoke condensate induces necrosis and apoptosis and inhibits cell growth in normal human bronchial epithelial cells
  
  Abstract summary: Both Quest CSC's are equally genotoxic, but have different cytotoxicity.
  
  Authors: Jinguo Chen, Defa Tian, Mohammad P. Valikhani, Alexandra Martin, Galina Holloway, Richard Higby, Rudolph J. Jaeger, Mike Johnson, Saijun Fan, and Peter G. Shields.
  
  Laboratory location: Georgetown University, Washington, DC and Arista Laboratories, Inc, Richmond, VA
  
  Date: : 2006 American Association for Cancer Research (AACR) Meeting
  
  

Steven G. Carmella, Menglan Chen, Shaomei Han, Joni Jensen, Anna Briggs, Dorothy K. Hatsukami, and Stephen S. Hecht

Masonic Cancer Center, University of Minnesota, USA

Abstract

Tobacco carcinogen and toxicant biomarkers, quantitative measurements of exposure and fate of specific tobacco carcinogens and toxicants, could in principle become an important part of a risk algorithm to identify smokers susceptible to lung cancer and are also important for the evaluation and potentially the regulation of new and existing tobacco products. Our goal is to develop a panel of tobacco carcinogen and toxicant biomarkers applicable in these activities. One validation criterion for a tobacco carcinogen or toxicant biomarker is its relationship to tobacco use. This can be determined by assessing the decrease in a biomarker level when people stop using the product. Therefore, in this study, we evaluated the relationship to cigarette smoking of eight tobacco carcinogen and toxicant biomarkers: 1-hydroxypyrene (1-HOP), a metabolite of pyrene and a widely accepted biomarker of exposure to carcinogenic polycyclic aromatic hydrocarbons; 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronides (total NNAL), an established biomarker of exposure to the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and 6 mercapturic acids (MA) derived from: 1,3 butadiene (MHBMA and DHBMA), acrolein (HPMA), crotonaldehyde (HBMA), benzene (SPMA), and ethylene oxide (HEMA) . Smokers provided 24h urine samples twice while still smoking and then on days 3, 7, 14, 21, 28, 42, and 56 after quitting. Seventeen subjects (11 female) completed the study. Their mean age (± S.D.) was 43.9 ± 11.0 years (range 23 - 58). Sixteen were Caucasian and one was of mixed racial heritage. They had been smoking an average of 17.3 ± 12.3 years and smoked 21.8 ± 6.7 cigarettes per day. MAs were quantified by a newly developed combined LC-negative ion-APCI-MS/MS method, total NNAL by GC-nitrosamine selective detection, and 1-HOP by LC-fluorescence. Baseline values of the biomarkers could be split into three groups: low, medium, and high. NNAL, 1-HOP, and SPMA comprised the low group (1-3 nmol/24h), HEMA and MHBMA the medium group (30 - 50 nmol/24h), and DHBMA, HPMA, and HBMA the high group (2600 - 11,000 nmol/24h). Levels of all MAs except DHBMA decreased rapidly after smoking cessation. In each case, the mean reduction of the MA from baseline was approximately 80% (P<0.0001 compared to baseline). Levels of DHBMA did not change significantly over the course of the study. Amounts of NNAL decreased gradually, with reductions of 55%, 72%, and 83% after 3, 7, and 21 days, respectively, consistent with previous studies. Levels of 1-HOP decreased to about 50% of the baseline values in most subjects. The results of this study demonstrate that, with the exception of DHBMA, the biomarkers quantified here are related to smoking and can be effectively used in studies of carcinogen and toxicant uptake in smokers.

Duanjun Tan, David S Goerlitz, Ramona G. Dumitrescu, Françoise Seillier-Moiseiwitsch, Stephanie M Spernak, Roy Anthony Orden, Jinguo Chen, Radoslav Goldman and Peter G. Shields*

Cancer Genetics and Epidemiology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA. 20057

Abstract

DNA alterations in mitochondria are believed to play a role in carcinogenesis, and are found in smoking-related cancers. We sought to replicate earlier findings for the association of smoking with increased mtDNA content in buccal cells, and further hypothesized that there would be an increased number of somatic mtDNA mutations in smokers. Buccal cells and blood lymphocytes were studied from 42 healthy smokers and 30 non-smokers. TTGE screening and sequencing was used to identify mtDNA mutations. The relative mtDNA content was determined by real-time PCR. Assuming that mtDNA in lymphocytes represent the inherited sequence, it was found that 31% of smokers harbored at least one somatic mtDNA mutation in buccal cells with a total of 39 point mutations and 8 short deletions/insertions. In contrast, only 23% of nonsmokers possessed mutations with a total of 10 point mutations and no insertions/deletions detected. MtDNA somatic mutation density was higher in smokers (0.68/10,000 bp/person) than in nonsmokers (0.2/10,000 bp/person). There was a statistically significant difference in the pattern of homoplasmy and heteroplasmy mutation changes between smokers and non-smokers. While nonsmokers had the most mutations in D-loop region (70%), smokers had mutations in both mRNA encoding gene (36%) and D-loop region (49%). The mean ratio of buccal cells to lymphocytes of mitochondrial DNA content in smokers was increased (2.81) when compared with nonsmokers (0.46). These results indicate that cigarette smoke affects mtDNA in buccal cells of smokers and so mtDNA damage might be a useful biomarker for smoke exposure, smoking-associated DNA damage, and cancer risk.

Jinguo Chen,* Higby Richard, † Defa Tian,* Duanjun Tan, * Michael Johnson,* Yingxian Xiao, * Kenneth J Kellar, * Shibao Feng, * Peter G. Shields *

*Lombardi Comprehensive Cancer Center, Georgetown University, Washington DC; † Arista Laboratories, Richmond, Virginia

Abstract

Low-nicotine and nicotine-free cigarettes are commercially available under the brand-name Quest®. Some consumers may believe that these are safer cigarettes, and they may smoke more cigarettes per day or inhale more smoke per cigarette to compensate for low nicotine yields. Thus, we have studied the toxicological effects of these two cigarettes and compared them with the Kentucky reference cigarette 2R4F. Also, the availability of nicotine-free cigarettes allows for the assessing the role of nicotine in cigarette smoke. Compared to the 2R4F, besides nicotine, some tobacco-specific nitrosamines, aldehydes, and volatile organic compounds were reduced in the Quest® cigarettes, while aromatic amines were higher in the nicotine-free compared with low nicotine cigarettes. The Salmonella reverse mutation assay revealed that cigarette smoke condensates from the nicotine-free (CSC-F), low nicotine (CSC-L) and 2R4F (CSC-R) cigarettes had a similar mutagenic potency. However, CSC-F had more inhibitory effects on the growth of normal human bronchial epithelial cells than CSC-L and CSC-R. Adding nicotine to the CSC-F attenuated this inhibition. Exposure to any CSC caused a similar dose-dependent LDH leakage. Both Quest® CSCs decreased gap junction intercellular communication. No CSC triggered apoptosis or induced expression of p53 and PARP proteins. Both Quest® CSCs caused cell cycle arrest. In conclusion, these studies are not consistent with a perception that low-nicotine or nicotine-free cigarettes may have less toxicity in human cells. While nicotine has anti-apoptotic properties, these studies indicate that nicotine, as it exists in CSC, attenuates cytotoxicity through non-apoptotic pathways.

Key words: cigarette smoke, nicotine, mutagenicity, cytotoxicity, apoptosis

DuanJun Tan, Jinguo Chen, David S Goerlitz, Ramona Dumitreascu, Roy Anthony Orden, Radoslav Goldman and Peter G Shields

Division of Cancer Genetics and Molecular Epidemiology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, 20057

Abstract

mtDNA is particularly susceptible to damage by mutagens. It exhibits 17 times higher rates of mutation than does the nuclear genome and DNA damage persists longer in the mitochondrial genome. DNA alterations in mitochondria are believed to play a role in carcinogenesis and are found in smoking-related cancers. Thus, we hypothesize that mtDNA damage would be a good biomarker of tobacco smoke exposure. We sought to replicate earlier findings for the association of smoking with increased mtDNA content in buccal cells, and further hypothesized that there would be an increased number of somatic mtDNA mutations in smokers. Buccal cells and blood lymphocytes were studied from 42 healthy smokers and 30 non-smokers. temporal temperature gradient electrophoresis screening and sequencing was used to identify mtDNA mutations. The relative mtDNA content was determined by real-time PCR. Assuming that mtDNA in lymphocytes represent the inherited sequence, it was found that 31% of smokers harbored at least one somatic mtDNA mutation in buccal cells with a total of 39 point mutations and 8 short deletions/insertions. In contrast, only 23% of nonsmokers possessed mutations with a total of 10 point mutations and no insertions/deletions detected. MtDNA somatic mutation density was higher in smokers (0.68/10,000 bp/person) than in nonsmokers (0.2/10,000 bp/person). There was a statistically significant difference in the pattern of homoplasmy and heteroplasmy mutation changes between smokers and non-smokers. While nonsmokers had the most mutations in D-loop region (70%), smokers had mutations in both mRNA encoding genes (36%) and D-loop region (49%). The mean buccal cell to lymphocyte ratio of mitochondrial DNA content in smokers was increased (2.81) compared to non-smokers (0.46). In addition, a low positive correlation between the ratio of buccal cell to lymphocyte mtDNA content and smoking status has been observed. These results suggest that cigarette smoke plays an important role in the increase in mtDNA mutations in the buccal cells of smokers. mtDNA mutations may be caused by specific mutagens in cigarette smoke in the mouth. The estimation of an individual's risk could be perhaps improved by coupling of mitochondrial mutations to other markers for tobacco smoke associated disease risk.

Jinguo Chen, Defa Tian, Mohammad P. Valikhani, Alexandra Martin, Galina Holloway, Richard Higby, Rudolph J. Jaeger, Mike Johnson, Saijun Fan, and Peter G. Shields.

Georgetown University, Washington, DC and Arista Laboratories, Inc, Richmond, VA

Abstract

Quest cigarettes are commercially available products that are sold in 3 forms, with 2 low levels of nicotine and one that is almost nicotine-free. They are advertised as a stepped-approach to reducing nicotine exposure. To characterize the toxicities of Quest cigarettes and the role of nicotine in it, we conducted a comparative evaluation of the genotoxicity and cytotoxicity of the mainstream cigarette smoke condensate (CSC) from Quest low-nicotine and nicotine-free cigarettes, in addition to the Kentucky reference cigarette K4R2F ("low tar" equivalent). As expected, the nicotine and NNK contents in the nicotine-free cigarette smoke (CS) were much less than low-nicotine CS or K4R2F cigarettes. Ames mutagenicity testing using the Salmonella bacterial strain TA98 indicated that there were no significant differences among the reference cigarette or the Quest cigarettes. Quest nicotine-free CSC (CSC-F) were more toxic to normal human bronchial epithelial (NHBE) cells than Quest low nicotine CSC (CSC-L), as evidenced by cell counting and WST-8 conversion, and nicotine added to CSC-F could increase cell survival, implying that nicotine in the CSC-L counteracts the tar cytotoxicity. Short-term exposure to both Quest CSC's resulted in a dose-dependent LDH leakage but there was no remarkable difference, indicating that they had similar necrotic effects on NHBE cells. In contrast, CSC-F caused more enrichment of nucleosomes in cytoplasma (apoptosis) than CSC-L. Furthermore, the nicotine could abrogate the induction of apoptosis by CSC-F, indicating the nicotine can inhibit the CSC-induced apoptosis. Study of cell death pathways revealed that p53 protein was weakly induced by CSC-F only in NHBE cells at the early passage and PARP expression was up-regulated in immortalized Beas-2B cells, not in NHBE cells. The proapoptotic protein Bax was found increase after exposure to CSC-F, but the expression of other apoptosis-related molecules such as Bcl-2, caspase 3, 9 did not alter or was not detected. Both CSC exposure also caused cell cycle arrest at G2/M checkpoint and CSC-F showed much arrest compared to CSC-L. However, nicotine alone did not affect the cell cycle progress, meaning that the cell cycle arrest was mediated by some compounds other than nicotine. In parallel, the cyclin D1 showed a significant decrease, indicating it may be involved in the cell cycle arrest. Taken together, these data demonstrate that both Quest CSC's are equally genotoxic, but have different cytotoxicity potentials in primary NHBE cells by inducing necrosis, apoptosis and causing cell cycle arrest. The apoptosis is mediated by Bax up-regulation, but independent of p53 pathways. CSC-F triggers more apoptosis than CSC-L because nicotine in the CSC-L inhibits the tar-induced apoptotic cell death and attenuates the cytotoxicity.