Differential cellular metabolite alterations in HaCaT cells caused by exposure to the aryl hydrocarbon receptor-binding polycyclic aromatic hydrocarbons chrysene, benzo[a]pyrene and dibenzo[a,l]pyrene

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the human environment. Since they are present in crude oilfractions used for the production of rubber and plastics, consumers may come into direct dermal contacts with these compounds (e.g., via tool handles) on a daily basis. Some individual PAHs are identified as genotoxic mutagens thereby prompting particular toxicological and environmental concern. Among this group, benzo[a]pyrene (BAP) constitutes a model carcinogen which is also used as reference compound for risk assessment purposes. It acts as a strong agonist of the aryl hydrocarbon receptor (AHR) and becomes metabolically activated toward mutagenic and carcinogenic intermediates by cytochrome P450-dependent monooxygenases (CYPs). While BAP has been exhaustively characterized with regard to its toxicological properties, there is much less information available for other PAHs. We treated an AHR-proficient immortal human keratinocyte cell line (i.e., HaCaT) with three selected PAHs: BAP, chrysene (CRY) and dibenzo[a,l]pyrene (DALP). Compound-mediated alterations of endogenous metabolites were investigated by an LC–MS/MS-based targeted approach. To examine AHR-dependent changes of the measured metabolites, AHR-deficient HaCaT knockdown cells (AHR-KD) were used for comparison. Our results reveal that 24 metabolites are sufficient to separate the PAH-exposed cells from untreated controls by application of a multivariate model. Alterations in the metabolomics profiles caused by each PAH show influences on the energy and lipid metabolism of the cells indicating reduced tricarboxylic acid (TCA) cycle activity and β-oxidation. Up-regulation of sphingomyelin levels after exposure to BAP and DALP point to pro-apoptotic processes caused by these two potent PAHs. Our results suggest that in vitro metabolomics can serve as tool to develop bioassays for application in hazard assessment. Keywords: Polycyclic aromatic hydrocarbons, Metabolomics, Aryl hydrocarbon receptor, Keratinocyte

Estrogenic Activity of Mineral Oil Aromatic Hydrocarbons Used in Printing Inks.

The majority of printing inks are based on mineral oils (MOs) which contain complex mixtures of saturated and aromatic hydrocarbons. Consumer exposure to these oils occurs either through direct skin contacts or, more frequently, as a result of MO migration into the contents of food packaging that was made from recycled newspaper. Despite this ubiquitous and frequent exposure little is known about the potential toxicological effects, particularly with regard to the aromatic MO fractions. From a toxicological point of view the huge amount of alkylated and unsubstituted compounds therein is reason for concern as they can harbor genotoxicants as well as potential endocrine disruptors. The aim of this study was to assess both the genotoxic and estrogenic potential of MOs used in printing inks. Mineral oils with various aromatic hydrocarbon contents were tested using a battery of in vitro assays selected to address various endpoints such as estrogen-dependent cell proliferation, activation of estrogen receptor α or transcriptional induction of estrogenic target genes. In addition, the comet assay has been applied to test for genotoxicity. Out of 15 MOs tested, 10 were found to potentially act as xenoestrogens. For most of the oils the effects were clearly triggered by constituents of the aromatic hydrocarbon fraction. From 5 oils tested in the comet assay, 2 showed slight genotoxicity. Altogether it appears that MOs used in printing inks are potential endocrine disruptors and should thus be assessed carefully to what extent they might contribute to the total estrogenic burden in humans

Estrogenic effects of MOs in the E-screen, the hERα-HeLa-9903 assay and induction of ER responsive transcripts as indicated.

<p>Proliferation assays with MCF-7 cells were performed subsequent to cellular stimulation with dispersions of 1 and 0.1 μl/ml MO (eq. to dil. of 1:1,000 and 1:10,000) or E2 as indicated (<b>A</b>). For the reporter gene assay hERα-HeLa-9903 cells were stimulated for 20 h with the indicated amounts of E2 or 1 μl/ml MO dispersed in medium, followed by cellular lysis and measurement of firefly luciferase activity (<b>B</b>). Transcriptional assays were following a 24-h exposure to 1 μl/ml MO or 10 nM E2, respectively (<b>C</b>). Data in all assays represent the mean ± SEM from at least three independent experiments. For the E-screen and the reporter gene assay data were corrected to accommodate the background of untreated cells and subjected to normalization using the effect of 1 nM E2. Likewise gene expression was normalized using <i>RPLP0</i> and the solvent control as references. Abbreviations: <i>PGR</i>, progesterone receptor (gene); <i>TFF1</i>, trefoil factor (gene); <i>GREB1</i>, estrogen-dependent growth regulator in breast cancer 1 (gene).</p

CYP induction and DNA damage caused by various MOs.

<p>Activation of AHR-dependent gene transcription in MCF-7 cells was measured following 24-h exposure to 1 μl/ml dispersed MO or 10 nM E2 (<b>A</b>). Data represent the mean ± SEM from at least three independent experiments. Gene expression levels were normalized to the reference gene <i>RPLP0</i> and the solvent control. For the comet assay (<b>B</b>), NHEKs were exposed to 1 μl/ml of dispersed MO, 3 μM benzo[<i>a</i>]pyrene (BP) or 20 μM methyl methanesulfonate (MMS) in presence of aphidicolin (APC). The resulting DNA damage was analyzed using an alkaline comet assay and quantified based on % DNA detected in the comet tails. Data represent the mean ± SEM of three independent experiments with cells from two individual donors resulting in 300 analyzed cells (* statistically significant (P<0.05) with respect to the APC/DMSO control as assessed by the Dunnett’s test). Abbreviations: <i>CYP1A1/1B1</i> (gene), cytochrome P450-dependent monooxygenase, family 1, subfamily A/B, polypeptide 1.</p

Analysis of the estrogenic potential of MOAHs.

<p>Activation of ER- (<b>A</b>) and AHR-dependent (<b>B</b>) gene transcription was measured following 24-h exposure to 1 μl/ml MOAHs or 10 nM E2, while luminescent reporter cells were subjected to 5 μl/ml MOAHs for 24 h (<b>C</b>). Data represent the mean ± SEM from at least three independent experiments. Again gene expression levels were normalized to the reference gene <i>RPLP0</i> and the solvent control, while luminescence values were corrected for the background activity of the solvent control and normalized using 5 nM E2 as maximal response. Blk (blank) represents the flow through of a mock sample which was treated similarly to the MOAH fractions. Abbreviations: <i>PGR</i>, progesterone receptor (gene); <i>TFF1</i>, trefoil factor (gene); <i>GREB1</i>, estrogen-dependent growth regulator in breast cancer 1 (gene); <i>CYP1A1/1B1</i> (gene), cytochrome P450-dependent monooxygenase, family 1, subfamily A/B, polypeptide 1.</p

Analytical separation and subsequent analysis of MOSH and MOAH fractions from MO 1.

<p>Transcriptional analysis of <i>CYP1A1</i> and <i>1B1</i> was used as a biological control to establish separation of MOSHs and MOAHs (<b>A</b>), while the E-screen (<b>B</b>) and estrogenic activity-dependent gene transcription (<b>C</b>) are readouts for xenoestrogenic potential. If not stated otherwise concentrations of E2 [M] and MO [μl/ml] were used as indicated or added as 10 nM E2. For the E-screen MOSH and MOAH fractions were added in final concentrations of 14.6 and 5.4 μg/ml, respectively, while the concentrations for the gene expression analysis were 146 μg/ml and 54 μg/ml, respectively. When fractions were diluted 1:100 for the transcriptional assays, additional positive and negative controls were applied to account for the final DMSO concentration (DMSO 1% and E2/DMSO). Relative gene expression was normalized to <i>RPLP0</i> as reference gene and to the solvent control. Graphs represent mean ± SEM from 3 independent experiments. Abbreviations: <i>PGR</i>, progesterone receptor (gene); <i>TFF1</i>, trefoil factor (gene); <i>GREB1</i>, estrogen-dependent growth regulator in breast cancer 1 (gene); <i>CYP1A1/1B1</i> (gene), cytochrome P450-dependent monooxygenase, family 1, subfamily A/B, polypeptide 1.</p