Cytochrome P450 1B1 mRNA levels in peripheral blood cells and exposure to polycyclic aromatic hydrocarbons in Chinese coke oven workers

doi:10.1016/S0048-9697(02)00070-0

Science of The Total Environment

Volume 296, Issues 1–3, 16 September 2002, Pages 27-33

https://doi.org/10.1016/S0048-9697(02)00070-0 Get rights and content

Abstract

Cytochrome P450 1B1 (CYP1B1) is induced through the Ah receptor and is involved in the activation of polycyclic aromatic hydrocarbons (PAHs). To determine the validity of a quantitative analysis of CYP1B1 mRNA in peripheral human blood cells for the estimation of PAH exposure, a real-time quantitative polymerase chain reaction method was used to measure the relative levels of CYP1B1 mRNA in 37 Chinese coke oven workers and 13 control workers. A large inter-individual difference in the levels was observed. The average level of the CYP1B1 mRNA in workers at the top work site, where the PAH exposure level from the coke ovens was highest, was significantly higher than in workers at the middle site (P<0.01) or the controls (P=0.02). A non-significant positive correlation was found between the CYP1B1 mRNA levels and urinary 1-hydroxypyrene (R=0.22, P=0.13), and a significant correlation between these mRNA levels and urinary cotinine (R=0.33, P=0.02). It was interesting that a significant positive correlation between CYP1B1 mRNA and 1-hydroxypyrene was observed in subjects with the Leu/Leu type of CYP1B1 Leu432Val polymorphism (R=0.33, P=0.02, n=38) and a non-significant correlation in subjects with the Leu/Val and Val/Val types (R=−0.36, P=0.25, n=12), although the number of subjects in this strata analysis was small. Our preliminary study suggests that PAH exposure in coke ovens and smoking maybe associated with CYP1B1 mRNA levels in peripheral blood cells although mRNA is generally unstable and could be expressed following exposure to other agents.

Introduction

Cytochrome P450 1B1 (CYP1B1) belongs to a multigene superfamily of monomeric mixed-function monooxygenases responsible for phase 1 metabolism (Sutter et al., 1994), and is involved in the activation of a broad spectrum of procarcinogens, especially polycyclic aromatic hydrocarbons (PAHs). CYP1B1 is induced by the Ah receptor (AhR) which is also characteristic of CYP1A1 (Tang et al., 1996).

CYP1B1 is expressed in many normal human tissues including peripheral blood cells (Sutter et al., 1994). Recently, Spencer et al. (Spencer et al., 1999) demonstrated a dose-dependent induction of CYP1B1 in peripheral lymphocytes by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in in vitro studies, and suggested the possibility of using CYP1B1 mRNA levels in peripheral blood cells as a marker of AhR ligands. However, few studies have explored the relationship between exposure to PAHs, known to be AhR ligands, and CYP1B1 mRNA levels in peripheral blood cells of subjects exposed to PAHs at high levels.

To determine the validity of a quantitative analysis of CYP1B1 mRNA in peripheral blood cells for the estimation of PAH exposure, this preliminary study analyzed CYP1B1 mRNA levels in Chinese coke oven workers exposed to PAHs at high levels using a real-time quantitative polymerase chain reaction.

Section snippets

Study population and subject selection

The subjects for this study were 37 coke oven workers and 13 controls who worked in the Angang Iron Steel Company, Anshang, China. They were from among 229 subjects in a cross-sectional study conducted in 1998–1999. The 229 subjects were selected from the top (high exposure), middle (medium exposure) and bottom (low exposure) work sites of coke oven plants, and control plants. These groups were matched for age and smoking status. The details of the selection method were similar to those of our

Results

Table 1 shows the basic characteristics and exposure indices of the 50 subjects in the coke ovens and the control plants. Mean age and smoking status were comparable in the two groups. Significant differences in urinary 1-OHP levels were observed between the coke workers and the controls (P<0.0001), although urinary cotinine levels were comparable between the two groups. The CBED, which was significantly related with 1-OHP (R=0.85, P<0.0001) and age (R=0.31, P=0.03), differed significantly

Discussion

Although the sample size of these study was relatively small, our observations suggest that CYP1B1 mRNA level in peripheral blood cells may be a marker of PAH exposure. The analyses were based on 50 subjects for whom sufficient cDNA specimens were obtained. Because of the low quantity of total RNA and cDNA, we excluded three-fourths of the subjects from our previous cross-sectional study. Although there was no difference in basic characteristics, including age, smoking status and the CYP1B1 leu

Acknowledgements

We are grateful to Hiroko Hashimoto and Tomoko Tokutake for their technical assistance. This work was supported in part by a Grant-in-Aid for the Second Term Comprehensive 10-Year Strategy for Cancer Control, and for Research on Environmental Health from the Ministry of Health and Welfare of Japan, and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan.

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Validation of gene expression profiles of candidate genes using low density array in peripheral blood of tobacco consuming head and neck cancer patients and auto/taxi drivers with preneoplastic lesions
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Several studies have also shown induction of CYP1A1, CYP1B1, and GSTM1 in peripheral blood lymphocytes isolated from individuals occupationally exposed to PAHs. This induction in the expression of CYPs was associated with the increase in urinary 1-OHP (PAHs exposure marker) which arise from traffic exhaust particles metabolized by the CYP1A1 metabolic pathway [27,28]. Increased oxidative DNA damage and chromosomal aberration reported in peripheral blood lymphocyte of traffic police officers and taxi drivers have also been attributed to PAHs exposure in these occupationally exposed individuals [29].
TaqMan Low-Density Array (TLDA) based Real-Time PCR (RT-PCR) of selected genes showed increased expression of polycyclic aromatic hydrocarbons (PAHs) metabolizing cytochrome P450s (CYPs), glutathione S-transferases (GSTs) and associated transcription factors in biopsy and peripheral blood samples isolated from head and neck squamous cell carcinoma (HNSCC) patients when compared to the controls. The genes involved in DNA repair, signal transduction pathway, EMT pathway, apoptosis, and cell adhesion/motility were found to be altered in both peripheral blood and biopsy samples of HNSCC patients. Transcription profiles in blood isolated from auto/taxi drivers, with pre-neoplastic lesions and history of tobacco use, also showed similar alterations. The present TLDA data thus demonstrates that low-density array of selected genes in peripheral blood has the potential to be used as a surrogate for providing insight into cancer progression pathways and possibly as an early biomarker for monitoring tobacco induced HNSCC.
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In the CYP1B1 gene, previous studies reported a significantly lower CYP1B1 mRNA expression in the rs1056836 CYP1B1*3/*3 homozygous genotype compared to CYP1B1*1/*1 and CYP1B1*3/*1 genotypes (Helmig et al., 2009). Additionally, a significant correlation between CYP1B1 mRNA expression and urinary 1-hydroxypyrene in coke oven workers with the CYP1B1*1/*1 wild-type genotype was described (Hanaoka et al., 2002). In concordance with these findings, our results suggest that the CYP1B1*3/*3 genotype conditioning poor PAH metabolism, is associated with significantly lower concentrations of DNA adducts in psoriatic patients than the remaining genotypes.
Goeckerman therapy (GT) for psoriasis combines the therapeutic effect of crude coal tar (CCT) and ultraviolet radiation (UVR). CCT contains polycyclic aromatic hydrocarbons, some of which can form DNA adducts that may induce mutations and contribute to carcinogenesis. The aim of our work was to evaluate the relationship between concentrations of benzo[a]pyrene-7,8-diol-9,10-epoxide-DNA adducts (BPDE-DNA adducts) and rs4646903 (CYP1A1 gene), rs1048943 (CYP1A1), rs1056836 (CYP1B1), rs1051740 (EPHX1), rs2234922 (EPHX1) and rs8175347 (UGT1A1) polymorphic sites, and GSTM1 null polymorphism in 46 patients with chronic stable plaque psoriasis who underwent GT. The level of BPDE-DNA adducts was determined using the OxiSelect BPDE-DNA Adduct ELISA Kit. Polymerase chain reaction (PCR) and restriction fragment length polymorphism analysis (rs4646903, rs1048943, rs1051740, and rs2234922), fragment analysis (rs8175347), real-time PCR (rs1056836), and digital droplet PCR polymorphism (GSTM1) were used. CYP1B1*1/*1 wild-type subjects and CYP1B1*3/*1 heterozygotes for rs1056836 formed significantly higher amounts of BPDE-DNA adducts than CYP1B1*3/*3 homozygotes (p = 0.031 and p = 0.005, respectively). Regarding rs1051740, individuals with EPHX1*3/*1 heterozygosity revealed fewer adducts than EPHX1*1/*1 wild-type subjects (p = 0.026). Our data suggest that CYP1B1/EPHX1 genotyping could help to predict the risk of DNA damage and to optimize doses of coal tar and UVR exposure in psoriatic patients in whom GT was applied.
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Polycyclic aromatic hydrocarbons (PAHs) are often detected in the environment and are regarded as endocrine disruptors. We here designated mixtures of PAHs in the environment as environmental PAHs (ePAHs) to discuss their effects collectively, which could be different from the sum of the constituent PAHs. We first summarized the biological impact of environmental PAHs (ePAHs) found in the atmosphere, sediments, soils, and water as a result of human activities, accidents, or natural phenomena. ePAHs are characterized by their sources and forms, followed by their biological effects and social impact, and bioassays that are used to investigate their biological effects. The findings of the bioassays have demonstrated that ePAHs have the ability to affect the endocrine systems of humans and animals. The pathways that mediate cell signaling for the endocrine disruptions induced by ePAHs and PAHs have also been summarized in order to obtain a clearer understanding of the mechanisms responsible for these effects without animal tests; they include specific signaling pathways (MAPK and other signaling pathways), regulatory mechanisms (chromatin/epigenetic regulation, cell cycle/DNA damage control, and cytoskeletal/adhesion regulation), and cell functions (apoptosis, autophagy, immune responses/inflammation, neurological responses, and development/differentiation) induced by specific PAHs, such as benz[a]anthracene, benzo[a]pyrene, benz[l]aceanthrylene, cyclopenta[c,d]pyrene, 7,12-dimethylbenz[a]anthracene, fluoranthene, fluorene, 3-methylcholanthrene, perylene, phenanthrene, and pyrene as well as their derivatives. Estrogen signaling is one of the most studied pathways associated with the endocrine-disrupting activities of PAHs, and involves estrogen receptors and aryl hydrocarbon receptors. However, some of the actions of PAHs are contradictory, complex, and unexplainable. Although several possibilities have been suggested, such as direct interactions between PAHs and receptors and the suppression of their activities through other pathways, the mechanisms underlying the activities of PAHs remain unclear. Thus, standardized assay protocols for pathway-based assessments are considered to be important to overcome these issues.
Differential relative effect potencies of some dioxin-like compounds in human peripheral blood lymphocytes and murine splenic cells
2014, Toxicology Letters
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As human peripheral blood is easy to collect, it is an interesting matrix for monitoring human health. Changes in AhR-mediated gene expressions in PBLs are widely used as biomarkers of human exposure to DLCs and polycyclic aromatic hydrocarbons (PAHs), despite the uncertainties and interindividual variability in their responses (Guida et al., 2013; Hanaoka et al., 2002; Hu et al., 2006; McHale et al., 2007; Van Duursen et al., 2005). Furthermore, present concerns regarding responses in human populations upon DLC exposure are more and more focused on extra-hepatic responses, including (neuro)development, reproductive functions, immunotoxicity and extra-hepatic carcinogenic responses (ECSCF, 2001; IARC, 2012; JECFA, 2001; Lauby-Secretan et al., 2013; UKCOT, 2001; USEPA, 2012).
Human risk assessment for dioxin-like compounds is typically based on the concentration measured in blood serum multiplied by their assigned toxic equivalency factor (TEF). Consequently, the actual value of the TEF is very important for accurate human risk assessment. In this study we investigated the effect potencies of three polychlorinated dibenzo-p-dioxins (PCDDs), six polychlorinated dibenzofurans (PCDFs) and 10 polychlorinated biphenyls (PCBs) relative to the reference congener 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) in in vitro exposed primary human peripheral blood lymphocytes (PBLs) and mouse splenic cells. REPs were determined based on cytochrome P450 (CYP) 1A1, 1B1 and aryl hydrocarbon receptor repressor (AhRR) gene expression as well as CYP1A1 activity in human PBLs and Cyp1a1 gene expression in murine splenic cells. Estimated median human REPs for 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (1234678-HpCDD), 2,3,4,7,8,-pentachlorodibenzofuran (23478-PeCDF), 1,2,3,4,7,8-hexachlorodibenzofuran (123478-HxCDF) and 1,2,3,4,7,8,9-heptachlorodibenzofuran (1234789-HpCDF) were with 0.1, 1.1, 1 and 0.09, respectively, significantly higher compared to those estimated for mouse with REPs of 0.05, 0.45, 0.09 and 0.04, respectively. Opposite to these results, the estimated median human REP of 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126), was with 0.001 30-fold lower compared to the mouse REP of 0.03. Furthermore, human REPs for 1234678-HpCDD, 23478-PeCDF, 123478-HxCDF, 1234789-HpCDF and PCB 126 were all outside the ± half log uncertainty range that is taken into account in the WHO-assigned TEFs. Together, these data show congener- and species-specific differences in REPs for some, but not all dioxin-like congeners tested. This suggests that, more emphasis should be placed on human-tissue derived REPs in the establishment of a TEF for human risk assessment.
Identification of cytochrome P450s involved in the metabolism of arachidonic acid in human platelets
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These differences in P450 expression levels may affect drug metabolism as well as endogenous substrate metabolism among different tissues. Some P450s, such as CYP3A4 and CYP1B1 [4,5], have been detected in human blood. Among blood cells, the CYP1A1, 2C9, 2C18, 2C19, 2J2, 2D6, 4A11 and 4F2 have been identified in lymphocytes and polynuclear leukocytes [6–8].
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Benzo[a]pyrene (B[a]P) is a small molecular weight carcinogen and the prototype of polycyclic aromatic hydrocarbons (PAHs). While these compounds are primarily known for their carcinogenicity, B[a]P and its metabolites are also toxic for mammalian immune cells.
To develop a prophylactic immune strategy against detrimental effects of B[a]P, we have immunized mice with a B[a]P-diphtheria toxoid conjugate vaccine. We showed that high levels of antibodies against B[a]P and its metabolites modulate the redistribution of these PAHs in the blood. After immunization, increased levels of B[a]P and its metabolites were recovered in the blood. B[a]P significantly suppressed the proliferative response of both T and B cells after a sub-acute administration, an effect that was completely reversed by vaccination. In immunized mice also the immunotoxic effect of B[a]P on IFN-γ, IL-12, TNF-α production and the reduced B cell activation was restored. Finally, our results showed that specific antibodies inhibited the induction of Cyp1a1 by B[a]P in lymphocytes and Cyp1b1 in the liver, enzymes that are known to convert the procarcinogen B[a]P to the ultimate DNA-adduct forming metabolite, a major risk factor of chemical carcinogenesis.
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Science of The Total Environment

Abstract

Introduction

Section snippets

Study population and subject selection

Results

Discussion

Acknowledgements

References (23)

Urinary 1-hydroxypyrene levels of garbage collectors with low-level exposure to polycyclic aromatic hydrocarbons

Sci Total Environ

Simple high-performance liquid chromatographic method for rapid determination of nicotine and cotinine in urine

J Chromatogr

Ah receptor ligands in tobacco smoke

Toxicol Lett

Diluted mainstream cigarette smoke condensates activate estrogen receptor and aryl hydrocarbon receptor-mediated gene transcription

Environ Res

Complete cDNA sequence of a human dioxin-inducible mRNA identifies a new gene subfamily of cytochrome P450 that maps to chromosome 2

J Biol Chem

Isolation and characterization of the human cytochrome P450 CYP1B1 gene

J Biol Chem

Association of cytochrome P450 1B1 (CYP1B1) polymorphism with steroid receptor status in breast cancer

Cancer Res

Expression of the CYP1A1 gene in peripheral lymphocytes as a marker of exposure to creosote in railroad workers

Cancer Epidemiol Biomarkers Prev

Cytochrome P450 1B1 mRNA measured in blood mononuclear cells by quantitative reverse transcription-PCR

Clin Chem

Detection of cytochrome P450 1B1 Bfr I polymorphism: genotype distribution in healthy German individuals and in patients with colorectal carcinoma

Pharmacogenetics

A novel method for real time quantitative RT-PCR

Genome Res

Cited by (33)

Validation of gene expression profiles of candidate genes using low density array in peripheral blood of tobacco consuming head and neck cancer patients and auto/taxi drivers with preneoplastic lesions

Genetic polymorphisms in biotransformation enzymes for benzo[a]pyrene and related levels of benzo[a]pyrene-7,8-diol-9,10-epoxide-DNA adducts in Goeckerman therapy

Biological impact of environmental polycyclic aromatic hydrocarbons (ePAHs) as endocrine disruptors

Differential relative effect potencies of some dioxin-like compounds in human peripheral blood lymphocytes and murine splenic cells

Identification of cytochrome P450s involved in the metabolism of arachidonic acid in human platelets

Modulation of Benzo[a]pyrene induced immunotoxicity in mice actively immunized with a B[a]P-diphtheria toxoid conjugate