Benzo[a]pyrene diol-epoxide DNA adducts and levels of polycyclic aromatic hydrocarbons in autoptic samples from human lungs

doi:10.1016/S0009-2797(98)00091-X

Chemico-Biological Interactions

Volume 116, Issue 3, 27 November 1998, Pages 199-212

https://doi.org/10.1016/S0009-2797(98)00091-X Get rights and content

Abstract

Benzo[a]pyrene (BaP) and other polycyclic aromatic hydrocarbons (PAHs) which are present in cigarette smoke, are common air and food genotoxic contaminants and possible human carcinogens. We measured the following PAH levels: benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, BaP, dibenzo[a,h]anthracene, benzo[g,h,i]perylene as well as (±) syn and anti BaP diol-epoxide (BPDE) DNA adducts in autopsy samples from the lungs of non-smokers, ex-smokers and smokers who had lived in Florence, Italy. PAH levels in lung tissue were similar in all groups, with the exception of dibenzo[a,h]anthracene (DBA), which was higher in lung samples from smokers (n=10, 0.18±0.17 ng/g d.w, mean ±S.D.) compared to non-smokers (n=15, 0.046±0.025 ng/g d.w) (P<0.05), whereas ex-smokers (n=5), had intermediate levels (0.07±0.03 ng/g d.w). The average level of total BPDE-DNA adducts was 4.46±5.76 per 10⁸ bases in smokers, 4.04±2.37 per 10⁸ in ex-smokers and 1.76±1.69 per 10⁸ in non-smokers. The levels of non-smokers were significantly different (P<0.05) from the levels of the smokers and ex-smokers combined. Total BPDE-DNA adducts were correlated with BaP levels in the lung samples in which both determinations were obtained (r=0.63). Our results demonstrate that the biological load of PAHs due to environmental pollution is similar in individuals who smoke and those who do not, but BPDE-DNA adducts are higher in smokers and ex-smokers compared to non-smokers. This study further confirms the usefulness of BPDE-DNA adduct levels determination in the lungs from autopsy samples for monitoring long-term human exposure to BaP, a representative PAH.

Introduction

Lung cancer is one of the leading causes of cancer-related death in many countries 1, 2, and cigarette smoke is considered to be one of its main determinants 3, 4, along with environmental pollution [5]. Since polycyclic aromatic hydrocarbons (PAHs) are potent mutagens, carcinogens and ubiquitous contaminants, they have been the object of extensive studies, but their contribution to the aetiology of human lung cancer either through exposure to polluted air [6]or smoking [7], remains controversial. PAHs, after metabolic activation, generate electrophilic species that covalently bind to DNA. In the case of benzo[a]pyrene (BaP), the formation of DNA adducts of BaP diol-epoxide (BPDE) is thought to play a role in the early stages of carcinogenesis, causing long-term genetic damage 8, 9, 10. Thus, these DNA adducts may be used as a biomarker of internal BaP exposure.

A number of studies have been published on the levels of aromatic DNA adducts in white blood cells using ³²P-postlabelling to determine BPDE-DNA adducts in smokers and non-smokers 11, 12, 13, 14, or in subjects professionally exposed to PAHs 15, 16, 17, 18, 19, 20. However the determination of aromatic DNA adducts in peripheral white blood cells and the attempt to find a correlation with lung carcinogenesis is questionable, since these cells have a short life span and are not the primary target for PAHs. On the contrary, the determination of BPDE-DNA adducts and of parent hydrocarbon levels in human lung samples is a more reliable indicator of the effective internal PAH dose. Therefore, the determination of DNA aromatic adduct levels in the target organ is, whenever possible, the most appropriate approach.

Accordingly, several studies have been published on total DNA aromatic adducts in human lung autoptic samples [21], and bronchial biopsies 22, 23, 24, using ³²P-postlabelling, an assay, which while reportedly more sensitive for detecting total DNA adduct levels, by itself does not allow the chemical identification of individual adducts.

Shields et al. [25]and Andreassen et al. [26], addressed this problem by coupling immunoaffinity chromatography with HPLC/synchronous fluorescence in order to detect specific BPDE-DNA adducts in the lungs of smokers and non-smokers. More recently improved HPLC/fluorometric assays (HPLC/FD) have been described 14, 27, 28, 29.

In this study we measured (±) syn and anti BPDE-DNA adducts by HPLC/FD and the levels of PAHs in autopsy samples from human lungs of non-professionally exposed subjects who had lived in Florence, Italy, in order to assess exposure to BaP and its correlation with BPDE-DNA adducts. The valley of Florence is characterised by a relatively uniform level of pollution from PAHs, mainly deriving from automotive emissions and wood combustion 30, 31.

Section snippets

Sample collection

Human lung specimens were obtained from autopsies performed by the Department of Pathology, University of Florence for routine microscopy. All samples were obtained from the same area of the lungs (apex of left inferior lobe). Following autopsy they were immediately frozen at −20°C in the Department of Pathology until analysis, which was completed within 30 days. Three lung samples were conserved and divided into three pieces to be analysed weekly in order to assess the relative stability of

Statistical analysis

Data were analysed with the Statgraphic Statistical Package (Manugistic, Cambridge, MD, USA) using linear regression analysis and one-way analysis of variance.

Results

Fig. 1 shows a chromatogram obtained from two standard BaP-tetrols (insert a); a sample of lung BPDE-DNA adducts obtained from a smoker (insert b) and from a non-smoker (insert c). The BaP-tetrols had a good chromatographic separation and no interfering peaks appeared.

PAH levels, determined in post-mortem lung tissues from non-smokers, ex-smokers and smokers were similar, with the exception of DBA which was significantly higher in smokers compared to non-smokers; ex-smokers had intermediate

Discussion

A number of papers 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, have been published on the levels of BPDE-adducts in white blood cells of individuals exposed to PAHs professionally and/or with cigarette smoke. They indicate in general that high exposure is associated with an increased level of aromatic adducts and with a high variability between groups and individuals. However, the determination of the level of adducts in peripheral blood cells, although certainly an indicator of exposure, does

Acknowledgements

This study was supported by funds of MURST 60%, Italy.

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Current risk assessments for environmental carcinogens rely on animal studies utilizing doses orders of magnitude higher than actual human exposures. Epidemiological studies of people with high exposures (e.g., occupational) are of value, but rely on uncertain exposure data. In addition, exposures are typically not to a single chemical but to mixtures, such as polycyclic aromatic hydrocarbons (PAHs). The extremely high sensitivity of accelerator mass spectrometry (AMS) allows for dosing humans with known carcinogens with de minimus risk. In this study UPLC-AMS was used to assess the toxicokinetics of [¹⁴C]-benzo[a]pyrene ([¹⁴C]-BaP) when dosed alone or in a binary mixture with phenanthrene (Phe). Plasma was collected for 48 h following a dose of [¹⁴C]-BaP (50 ng, 5.4 nCi) or the same dose of [¹⁴C]-BaP plus Phe (1250 ng). Following the binary mixture, C_max of [¹⁴C]-BaP significantly decreased (4.4-fold) whereas the volume of distribution (V_d) increased (2-fold). Further, the toxicokinetics of twelve [¹⁴C]-BaP metabolites provided evidence of little change in the metabolite profile of [¹⁴C]-BaP and the pattern was overall reduction consistent with reduced absorption (decrease in C_max). Although Phe was shown to be a competitive inhibitor of the major hepatic cytochrome P-450 (CYP) responsible for metabolism of [¹⁴C]-BaP, CYP1A2, the high inhibition constant (K_i) and lack of any increase in unmetabolized [¹⁴C]-BaP in plasma makes this mechanism unlikely to be responsible. Rather, co-administration of Phe reduces the absorption of [¹⁴C]-BaP through a mechanism yet to be determined. This is the first study to provide evidence that, at actual environmental levels of exposure, the toxicokinetics of [¹⁴C]-BaP in humans is markedly altered by the presence of a second PAH, Phe, a common component of environmental PAH mixtures.
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This list of 16 US - EPA PAHs plays an extremely important role as a regularized set of chemical compounds to be analyzed, especially in environmental investigations; however, this list has never been updated. A substantial amount of information has been accumulated on the carcinogenic and mutagenic potential of the 16 US - EPA priority PAHs using different experimental models (Ke et al., 2018; Krishnamurthi et al., 2006; Lodovici et al., 1998; Luckert et al., 2013; Stout et al., 2015; Wang et al., 2015; White, 2002; Yan et al., 2004). However, several years of research on the toxic effects of other PAHs cannot be overlooked.
Knowledge of the toxic potential of polycyclic aromatic hydrocarbons (PAHs) has increased over time. Much of this knowledge is about the 16 United States - Environmental Protection Agency (US - EPA) priority PAHs; however, there are other US – EPA non-priority PAHs in the environment, whose toxic potential is underestimated. We conducted a systematic review of in vitro, in vivo, and in silico studies to assess the genotoxicity, mutagenicity, and carcinogenicity of 13 US - EPA non-priority parental PAHs present in the environment. Electronic databases, such as Science Direct, PubMed, Scopus, Google Scholar, and Web of Science, were used to search for research with selected terms without time restrictions. After analysis, following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) protocol, 249 articles, published between 1946 and 2020, were selected and the quality assessment of these studies was performed. The results showed that 5-methylchrysene (5-MC), 7,12-dimethylbenz[a]anthracene (7,12-DMBA), cyclopenta[cd]pyrene (CPP), and dibenzo[al]pyrene (Db[al]P) were the most studied PAHs. Moreover, 5-MC, 7,12-DMBA, benz[j]aceanthrylene (B[j]A), CPP, anthanthrene (ANT), dibenzo[ae]pyrene (Db[ae]P), and Db[al]P have been reported to cause mutagenic effects and have been being associated with a risk of carcinogenicity. Retene (RET) and benzo[c]fluorene (B[c]F), the least studied compounds, showed evidence of a strong influence on the mutagenicity and carcinogenicity endpoints. Overall, this systematic review provided evidence of the genotoxic, mutagenic, and carcinogenic endpoints of US - EPA non-priority PAHs. However, further studies are needed to improve the future protocols of environmental analysis and risk assessment in severely exposed populations.
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The analysis of polycyclic aromatic hydrocarbon (PAH) in human lungs is a challenge, due to low amount of target analytes, low amount of available sample and complexity of the matrix. In this study, a micro-scale analytical method is proposed to determine PAH in human lungs. The method employs micro-scale extraction, clean-up, and programmable temperature vaporisation-gas chromatography-mass spectrometry (PTV-GC–MS) analysis. The sample treatment was minimized. PTV was an excellent option to introduce up to 50 µL with a variation of <10%. Determination coefficients (r²) ranged from 0.967 for acenaphthene to 0.996 for indeno[1,2,3-cd]pyrene (p < 0.05). Instrumental detection limits were between 100 fg µL⁻¹ (benzo[a]pyrene) and 5 pg μL⁻¹, for acenaphthylene; the method detection limits were between 100 pg g⁻¹ (indeno[1,2,3-cd]pyrene) and 6.7 ng g⁻¹ (pyrene). Recovery ranged from 46 ± 12% (indeno[1,2,3-cd]pyrene) to 79 ± 16% (benzo[e]pyrene) with ≤5 ng of PAH, and between 82 ± 15% (acenaphthylene) and 98 ± 7% (benzo[k]fluoranthene) with ~10 ng of PAH in the lungs. The Analytical Eco-Scale metric classified our methodology as an acceptable green analysis. The analytical method was applied to determine PAH in real lung samples. No PAH were found in any sample. Cholesterol and derivatives, in addition to palmitic and oleic acid, were the most abundant compounds. Our proposal is a microscale, green and sensitive method to determine PAH levels in human lung tissues.
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Somewhat surprisingly, history of exposure (smoking, occupational) has been reported as not significantly elevating BaP-DHDE-DNA adducts in PBMCs or other tissues (Boysen and Hecht, 2003; Carstensen et al., 1999). Other studies report modestly higher levels in smoker's compared to non- or ex-smokers (Lodovici et al., 1998; Villalta et al., 2017) Examination of 10 human lung biopsy samples by Monein et al. (2015) found no detectable (LOD 1–3 adducts per 108 nucleotides) BaP-DHDE-dG adducts. Recent advances in methodology, leading to increased specificity and sensitivity for assaying BaP-DNA adducts, have improved (LOD of ~ 1 adduct in 1011 nucleotides).
Benzo[a]pyrene (BaP), is a known human carcinogen (International Agency for Research on Cancer (IARC) class 1). The remarkable sensitivity (zepto-attomole ¹⁴C in biological samples) of accelerator mass spectrometry (AMS) makes possible, with de minimus risk, pharmacokinetic (PK) analysis following [¹⁴C]-BaP micro-dosing of humans. A 46 ng (5 nCi) dose was given thrice to 5 volunteers with minimum 2 weeks between dosing and plasma collected over 72 h. [¹⁴C]-BaP_eq PK analysis gave plasma T_max and C_max values of 1.25 h and 29–82 fg/mL, respectively. PK parameters were assessed by non- compartment and compartment models. Intervals between dosing ranged from 20 to 420 days and had little impact on intra-individual variation. DNA, extracted from peripheral blood mononuclear cells (PBMCs) of 4 volunteers, showed measurable levels (LOD ~ 0.5 adducts/10¹¹ nucleotides) in two individuals 2–3 h post-dose, approximately three orders of magnitude lower than smokers or occupationally-exposed individuals. Little or no DNA binding was detectable at 48–72 h. In volunteers the allelic variants CYP1B1^*1/*⁎1, ^*1/*3 or ^*3/*3 and GSTM1^*^0/0 or ^*1 had no impact on [¹⁴C]-BaP_eq PK or DNA adduction with this very limited sample. Plasma metabolites over 72 h from two individuals (one CYP1B1^*1/*1 and one CYP1B1^*3/*3) were analyzed by UPLC-AMS. In both individuals, parent [¹⁴C]-BaP was a minor constituent even at the earliest time points and metabolite profiles markedly distinct. AMS, coupled with UPLC, could be used in humans to enhance the accuracy of pharmacokinetics, toxicokinetics and risk assessment of environmental carcinogens.

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Benzo[a]pyrene diol-epoxide DNA adducts and levels of polycyclic aromatic hydrocarbons in autoptic samples from human lungs

Abstract

Introduction

Section snippets

Sample collection

Statistical analysis

Results

Discussion

Acknowledgements

Mayo Clin. Proc.

Toxicology

Mutat. Res.

Toxic. Lett.

Chem. Biol. Interact.

Mutat. Res.

Sci. Total Environ.

Chemosphere

Biochem. Biophys. Res. Comm.

Mutat. Res.

Mutat. Res.

Fifty years of BaP

Nature

Polycyclic aromatic hydrocarbons in the occupational work environment

Scand. J. Work Environ. Health

Tumour multiplicity, DNA adducts and K-ras mutation pattern of 5-methylchrysene in strain A/J mouse lung

Carcinogenesis

Adenomas induced by polycyclic aromatic hydrocarbons in strain A/J mouse lung correlate with time-integrated DNA adduct levels

Cancer Res.