Influence of data acquisition modes and data analysis approaches on non-targeted analysis of phthalate metabolites in human urine.

Humans are often exposed to phthalates and their alternatives, on account of their widespread use in PVC as plasticizers, which are associated with harmful human effects. While targeted biomonitoring provides quantitative information for exposure assessment, only a small portion of phthalate metabolites has been targeted. This results in a knowledge gap in human exposure to other unknown phthalate compounds and their metabolites. Although the non-targeted analysis (NTA) approach is capable of screening a broad spectrum of chemicals, there is a lack of harmonized workflow in NTA to generate reproducible data within and between different laboratories. The objective of this study was to compare two different NTA data acquisition modes, the data-dependent (DDA) and independent (DIA) acquisition (DDA), as well as two data analysis approaches, based on diagnostic ions and Compound Discoverer software for the prioritization of candidate precursors and identification of unknown compounds in human urine. Liquid chromatography coupled to high-resolution mass spectrometry was used for sample analysis. The combination of three-diagnostic-ion extraction and DDA data acquisition was able to improve data filtering and data analysis for prioritizing phthalate metabolites. With DIA, 25 molecular features were identified in human urine, while 32 molecular features were identified in the same urine samples using DDA data. The number of molecular features identified with level 1 confidence was 11 and 9 using DIA and DDA data, respectively. The study demonstrated that besides sample preparation, the impact of data acquisition must be taken into account when developing a NTA method and a consistent protocol for evaluating such an impact is necessary.

Exploring chemical space in non-targeted analysis: a proposed ChemSpace tool.

Non-targeted analysis (NTA) using high-resolution mass spectrometry allows scientists to detect and identify a broad range of compounds in diverse matrices for monitoring exposure and toxicological evaluation without a priori chemical knowledge. NTA methods present an opportunity to describe the constituents of a sample across a multidimensional swath of chemical properties, referred to as "chemical space." Understanding and communicating which region of chemical space is extractable and detectable by an NTA workflow, however, remains challenging and non-standardized. For example, many sample processing and data analysis steps influence the types of chemicals that can be detected and identified. Accordingly, it is challenging to assess whether analyte non-detection in an NTA study indicates true absence in a sample (above a detection limit) or is a false negative driven by workflow limitations. Here, we describe the need for accessible approaches that enable chemical space mapping in NTA studies, propose a tool to address this need, and highlight the different ways in which it could be implemented in NTA workflows. We identify a suite of existing predictive and analytical tools that can be used in combination to generate scores that describe the likelihood a compound will be detected and identified by a given NTA workflow based on the predicted chemical space of that workflow. Higher scores correspond to a higher likelihood of compound detection and identification in a given workflow (based on sample extraction, data acquisition, and data analysis parameters). Lower scores indicate a lower probability of detection, even if the compound is truly present in the samples of interest. Understanding the constraints of NTA workflows can be useful for stakeholders when results from NTA studies are used in real-world applications and for NTA researchers working to improve their workflow performance. The hypothetical ChemSpaceTool suggested herein could be used in both a prospective and retrospective sense. Prospectively, the tool can be used to further curate screening libraries and set identification thresholds. Retrospectively, false detections can be filtered by the plausibility of the compound identification by the selected NTA method, increasing the confidence of unknown identifications. Lastly, this work highlights the chemometric needs to make such a tool robust and usable across a wide range of NTA disciplines and invites others who are working on various models to participate in the development of the ChemSpaceTool. Ultimately, the development of a chemical space mapping tool strives to enable further standardization of NTA by improving method transparency and communication around false detection rates, thus allowing for more direct method comparisons between studies and improved reproducibility. This, in turn, is expected to promote further widespread applications of NTA beyond research-oriented settings.

An Introduction to the Benchmarking and Publications for Non-Targeted Analysis Working Group.

Non-targeted analysis (NTA) encompasses a rapidly evolving set of mass spectrometry techniques aimed at characterizing the chemical composition of complex samples, identifying unknown compounds, and/or classifying samples, without prior knowledge regarding the chemical content of the samples. Recent advances in NTA are the result of improved and more accessible instrumentation for data generation and analysis tools for data evaluation and interpretation. As researchers continue to develop NTA approaches in various scientific fields, there is a growing need to identify, disseminate, and adopt community-wide method reporting guidelines. In 2018, NTA researchers formed the Benchmarking and Publications for Non-Targeted Analysis Working Group (BP4NTA) to address this need. Consisting of participants from around the world and representing fields ranging from environmental science and food chemistry to 'omics and toxicology, BP4NTA provides resources addressing a variety of challenges associated with NTA. Thus far, BP4NTA group members have aimed to establish a consensus on NTA-related terms and concepts and to create consistency in reporting practices by providing resources on a public Web site, including consensus definitions, reference content, and lists of available tools. Moving forward, BP4NTA will provide a setting for NTA researchers to continue discussing emerging challenges and contribute to additional harmonization efforts.

TBAI-catalyzed selective synthesis of sulfonamides and ß-aryl sulfonyl enamines: coupling of arenesulfonyl chlorides and sodium sulfinates with tert-amines.

A simple, practical and metal-free method has been developed for the synthesis of sulfonamides and ß-arylsulfonyl enamines via the selective cleavage of C-N and C-H bonds through the iodine-catalyzed oxidation of arenesulfonyl chlorides and sodium sulfinates with tert-amines. The method uses commercially available inexpensive catalysts and oxidants, and has a wide substrate scope and operational simplicity.

Bisphenol Analogues Other Than BPA: Environmental Occurrence, Human Exposure, and Toxicity-A Review.

Numerous studies have investigated the environmental occurrence, human exposure, and toxicity of bisphenol A (BPA). Following stringent regulations on the production and usage of BPA, several bisphenol analogues have been produced as a replacement for BPA in various applications. The present review outlines the current state of knowledge on the occurrence of bisphenol analogues (other than BPA) in the environment, consumer products and foodstuffs, human exposure and biomonitoring, and toxicity. Whereas BPA was still the major bisphenol analogue found in most environmental monitoring studies, BPF and BPS were also frequently detected. Elevated concentrations of BPAF, BPF, and BPS (i.e., similar to or greater than that of BPA) have been reported in the abiotic environment and human urine from some regions. Many analogues exhibit endocrine disrupting effects, cytotoxicity, genotoxicity, reproductive toxicity, dioxin-like effects, and neurotoxicity in laboratory studies. BPAF, BPB, BPF, and BPS have been shown to exhibit estrogenic and/or antiandrogenic activities similar to or even greater than that of BPA. Knowledge gaps and research needs have been identified, which include the elucidation of environmental occurrences, persistence, and fate of bisphenol analogues (other than BPA), sources and pathways for human exposure, effects on reproductive systems and the mammary gland, mechanisms of toxicity from coexposure to multiple analogues, metabolic pathways and products, and the impact of metabolic modification on toxicity.

Expanding the scope of pressure-assisted electrokinetic injection for online concentration of positively charged analytes in capillary electrophoresis-mass spectrometry.

Sample injection is a crucial step in CE. In past, many efforts have been focused on concentrating the analytes into a sharp sample plug. In 2006, pressure-assisted electrokinetic injection (PAEKI) was proposed as a new preconcentration technique for anions. In this study, we expanded the applicability of PAEKI to online preconcentrate positively charged analytes. L-Arginine, L-lysine, and imidazole were chosen as target analytes for method development. The enhancement factor of PAEKI was over 3000-fold. When CZE was coupled with a Q-TOF system, PAEKI delivers a detection limit of 18-28 pg/mL and a dynamic calibration range over four orders of magnitude. The RSD was less than 6.4% (n = 4) on both peak area and migration time, indicating a good repeatability.

In vitro exposure to cigarette smoke induces oxidative stress in follicular cells of F1 hybrid mice.

This study assessed the influence of cigarette smoke condensate (CSC) and benzo(a)pyrene [B(a)P] on the levels of two oxidative stress biomarkers [8-isoprostane (8-IsoP) and 8-hydroxy-2-deoxy Guanosine (8-OH-dG)], in in-vitro spent media of follicle cells. Follicles (100-130 µm) isolated from ovaries of F1 hybrid (C57Bl/6j × CBA/Ca) mice were cultured for 13 days in media exposed to B(a)P [0 ng ml⁻¹ (control) to 45 ng ml⁻¹] or CSC [0 µg ml⁻¹ (control) to 130 µg ml⁻¹]. The concentrations of oxidative stress biomarkers in spent media were quantified by enzyme-linked immune sorbent assays (ELISA). CSC and B(a)P treatment induced a significant, dose-dependent increase in the concentrations of 8-IsoP and 8-OH-dG in the spent media. We conclude that CSC and B(a)P exposure can induce oxidative stress in ovarian follicles, an effect that may contribute to the previously documented decline in follicle development and premature ovarian failure in women who smoke.

Development of a robust non-targeted analysis approach for fast identification of endocrine disruptors and their metabolites in human urine for exposure assessment.

Endocrine disrupting chemicals are of concern because of possible human health effects, thus they are frequently included in biomonitoring studies. Current analytical methods are focused on known chemicals and are incapable of identifying or quantifying other unknown chemicals and their metabolites. Non-targeted analysis (NTA) methods are advantageous since they allow for broad chemical screening, which provides a more comprehensive characterization of human chemical exposure, and can allow elucidation of metabolic pathways for unknown chemicals. There are still many challenges associated with NTA, which can impact the results obtained. The chemical space, i.e., the group of known and possible compounds within the scope of the method, must clearly be defined based on the sample preparation, as this is critical in identifying chemicals with confidence. Data acquisition modes and mobile phase additives used with liquid chromatography coupled to high-resolution mass-spectrometry can affect the chemicals ionized and structural identification based on the spectral quality. In this study, a sample preparation method was developed using a novel clean-up approach with CarbonS cartridges, for endocrine-disrupting chemicals in urine, including new bisphenol A analogues and benzophenone-based UV filters, like methyl bis (4-hydroxyphenyl acetate). The study showed that data dependent acquisition (DDA) had a lower identification rate (40%) at low spiking levels, i.e., 1 ng/mL, compared to data independent acquisition (DIA) (57%), when Compound Discoverer was used. In DDA, more compounds were identified using Compound Discoverer, with an identification rate of 95% when ammonium acetate was compared to acetic acid (82%) as a mobile phase additive. TraceFinder software had an identification rate of 53% at 1 ng/mL spiking level using the DDA data, compared to 40% using the DIA data. Using the developed method, 2,4 bisphenol F was identified for the first time in urine samples. The results show how NTA can provide human exposure information for risk assessment and regulatory action but standardized reporting of procedures is needed to ensure study results are reproducible and accurate. His Majesty the King in Right of Canada, as represented by the Minister of Health, 2024.

Determination of isoflavones in rat serum using liquid chromatography-tandem mass spectrometry with a highly efficient core-shell column.

Consumption and nutritional supplementation of soy and soy-based products have been linked to health benefits such as lower cholesterol and triglyceride levels, and decreased incidence of cardiovascular disease and diabetes. In this study, we have developed a sensitive, specific, and robust method using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for determination of serum isoflavones. A new highly efficient pentafluorophenyl phase core-shell column was first used to separate all isoflavones within 3 min, a separation time which is comparable to ultra-pressure liquid chromatography (UPLC) and micro-HPLC. A two-enzyme hydrolysis system with sulfatase and ß-glucuronidase has also been developed to improve the efficiency of deconjugation of conjugated isoflavones in serum. The corresponding conjugated isoflavones were used to evaluate recoveries. In addition to duplicates, the method of standard addition was also applied in sample analysis for quality control. The developed method was applied to the analysis of 32 serum samples and was shown to be specific, sensitive and reproducible.

Intermediate volatile organic compounds in Canadian residential air in winter: Implication to indoor air quality.

Intermediate volatile organic compounds (IVOCs) have recently been characterized for their contributions to the formation of secondary organic aerosol in atmospheric air. However, IVOCs in air in various indoor environments have not been characterized yet. In this study, we characterized and measured IVOCs, volatile organic compounds (VOCs) and semi-volatile organic compounds (SVOCs), in residential indoor air in Ottawa, Canada. IVOCs, including n-alkanes, branched-chain alkanes (b-alkanes), unspecified complex mixtures (UCM) IVOCs, and oxygenated IVOCs (such as fatty acids), were found to have a large impact on indoor air quality. The results indicate that the indoor IVOCs behave differently from those in the outdoor environment. IVOCs in the studied residential air ranged from 14.4 to 69.0 µg/m3, with a geometric mean of 31.3 µg/m3, accounting for approximately 20% of the total organic compounds (IVOCs, VOCs and SVOCs) in indoor air. The total b-alkanes and UCM-IVOCs were found to have statistically significant positive correlations with indoor temperature but have no correlations with airborne particulate matter less than 2.5 µm (PM2.5) as well as ozone (O3) concentration. However, indoor oxygenated IVOCs behaved differently from b-alkanes and UCM-IVOCs, with a statistically significant positive correlation with indoor relative humidity but no correlation with other indoor environmental conditions.

Non-targeted analysis (NTA) and suspect screening analysis (SSA): a review of examining the chemical exposome.

Non-targeted analysis (NTA) and suspect screening analysis (SSA) are powerful techniques that rely on high-resolution mass spectrometry (HRMS) and computational tools to detect and identify unknown or suspected chemicals in the exposome. Fully understanding the chemical exposome requires characterization of both environmental media and human specimens. As such, we conducted a review to examine the use of different NTA and SSA methods in various exposure media and human samples, including the results and chemicals detected. The literature review was conducted by searching literature databases, such as PubMed and Web of Science, for keywords, such as "non-targeted analysis", "suspect screening analysis" and the exposure media. Sources of human exposure to environmental chemicals discussed in this review include water, air, soil/sediment, dust, and food and consumer products. The use of NTA for exposure discovery in human biospecimen is also reviewed. The chemical space that has been captured using NTA varies by media analyzed and analytical platform. In each media the chemicals that were frequently detected using NTA were: per- and polyfluoroalkyl substances (PFAS) and pharmaceuticals in water, pesticides and polyaromatic hydrocarbons (PAHs) in soil and sediment, volatile and semi-volatile organic compounds in air, flame retardants in dust, plasticizers in consumer products, and plasticizers, pesticides, and halogenated compounds in human samples. Some studies reviewed herein used both liquid chromatography (LC) and gas chromatography (GC) HRMS to increase the detected chemical space (16%); however, the majority (51%) only used LC-HRMS and fewer used GC-HRMS (32%). Finally, we identify knowledge and technology gaps that must be overcome to fully assess potential chemical exposures using NTA. Understanding the chemical space is essential to identifying and prioritizing gaps in our understanding of exposure sources and prior exposures. IMPACT STATEMENT: This review examines the results and chemicals detected by analyzing exposure media and human samples using high-resolution mass spectrometry based non-targeted analysis (NTA) and suspect screening analysis (SSA).

Diagnostic Fragmentation Pathways for Identification of Phthalate Metabolites in Nontargeted Analysis Studies.

Phthalates have been studied due to their linkages with adverse developmental effects; however, metabolites of this class of compounds are undercharacterized and are poorly captured by traditional targeted analysis. In this study, we developed a nontargeted analysis approach for identifying and classifying phthalate metabolites based on a comprehensive study of their fragmentation pathways in electrospray ionization (ESI) quadrupole-time-of-flight mass spectrometry (QTOF-MS). This approach identifies molecular features in the data as phthalate metabolites via the detection of three structurally significant fragment ions. Then phthalate metabolites are classified into four types based on the presence of additional fragment ions specific to each type. Cleavage mechanisms for each class of phthalate metabolite are proposed based on fragmentation patterns generated at various collision energies (CE). All of the tested phthalate metabolites including oxidative and nonoxidative metabolites produced a fragment ion at m/z 121.0295, representing the deprotonated benzoate ion [C6H5COO]-. Most tested phthalate metabolites can produce a specific ion at m/z 147.0088, the deprotonated o-phthalic anhydride ion. However, phthalate carboxylate metabolites can only produce the [M-H-R]- ion at m/z 165.0193 and do not produce the fragment at m/z 147.0088. Other phthalate oxidative metabolites (hydroxyl- and oxo-) follow a different fragmentation pathway than nonoxidative metabolites. With this workflow, eight unknown phthalate metabolites were putatively identified in pooled urine, with one identified as a previously unreported metabolite by a combination of the MS/MS spectrum and the predicted retention time. Method detection limits for phthalate metabolites in urine were also estimated.

Use of reference chemicals to determine passive uptake rates of common indoor air VOCs by collocation deployment of active and passive samplers.

Passive samplers have become more popular in their application in the measurement of airborne chemicals. For volatile organic compounds, the rate of a chemical's diffusivity is a determining factor in the quantity of the chemical being collected for a given passive sampler. While uptake rate of a chemical in the passive sampler can be determined either by collocation deployment of both active and passive samplers or use of controlled facilities such as environmental chambers, a new approach without a need for accurate active flow rate in the collocation experiment was demonstrated in this study. This approach uses chemicals of known uptake rates as references to calculate the actual flow rate of the active sampling in the collocation experiment. The active sampling rate in turn can be used in the determination of the uptake rates of all other chemicals present in the passive samplers. The advantage of such approach is the elimination of the errors in actual active sampling rate associated with low flow employed in the collocation experiment. Using this approach, passive uptake rates of more than 80 volatile organic compounds commonly present in indoor air were determined. These experimentally determined uptake rates correlate well with air diffusivity of the chemicals, indicating the regression equation describing such correlation might be useful in predicting the uptake rates of other volatile organic chemicals in indoor air based on their air diffusivity.

A tool for rapid screening of direct DNA agents using reaction rates and relative interaction potency: towards screening environmental contaminants for hazard.

DNA damage represents a potential biomarker for determining the exposure risk to chemicals and may provide early warning data for identifying chemical hazards to human health. Here, we have demonstrated a simple chromatography-based method that can be used to rapidly screen for the presence of chemical hazards as well as to determine parameters relevant to hazard assessment. In this proof-of-principle study, a simple in vitro system was used to determine the interaction of pollutants and probable carcinogens, phenyl glycidyl ether (PGE), tetrachlorohydroquinone (Cl(4)HQ), methylmethane sulfonate (MMS), styrene-7,8-oxide (SO), and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), a metabolite of benzo[a]pyrene (B[a]P), with single- and double-stranded DNA probes. Differences in potency and reaction kinetics were studied for chemical and DNA type. A relative interaction potency equivalency (PEQ) of a chemical was determined by ratio of interaction potency of a chemical to BPDE as the reference chemical in the reaction with single- and double-stranded oligodeoxynucleotides. PEQs were found to be BPDE > PGE > SO > MMS > Cl(4)HQ for single-stranded oligodeoxynucleotides while they were found to be BPDE > PGE > Cl(4)HQ > MMS > SO for double-stranded oligodeoxynucleotides. Kinetics evaluation revealed that BPDE reacted with both DNA probes at a significantly faster rate, as compared to the remaining test chemicals. Equilibrium was reached within an hour for BPDE, but required a minimum of 48 h for the remaining chemicals. First-order rate constants were (1.61 ± 0.2) × 10(-3) s(-1) and (3.18 ± 0.4) × 10(-4) s(-1) for reaction of BPDE with double- and single-stranded DNA, respectively. The remaining chemicals possessed rate constants from 2 to 13 × 10(-6) s(-1) with a relative kinetic order for reaction with DNA of BPDE ≫ MMS > SO > PGE > Cl(4)HQ for ds-DNA and BPDE ≫ SO ≈ Cl(4)HQ ≈ MMS > PGE for ss-DNA. We further found that the reaction potency, defined by dose-response between chemical pollutants and DNA, depends on the form of DNA present for reaction. Noteworthy, we found that relative PEQ did not follow the same kinetic trends. However, our preliminary findings suggest that reaction kinetics, in combination with relative interaction potency, may be a significant parameter that can be used to evaluate the hazard level of environmental pollutants.

Prediction of liquid chromatographic retention time using quantitative structure-retention relationships to assist non-targeted identification of unknown metabolites of phthalates in human urine with high-resolution mass spectrometry.

The non-targeted analysis and identification of contaminant metabolites such as metabolites of phthalates and their alternatives in human biofluid samples constitutes a growing research field in human biomonitoring because of their importance as biomarkers of human exposure to the parent compounds. High-resolution mass spectrometry (HRMS) combined with high-performance liquid chromatography (HPLC) can provide fast separation and sensitive analysis using this application. However, the diversity of potential metabolites, especially isomers, in human samples, makes mass spectrometry-based structural identification very challenging, even with high-resolution and accurate mass. In this study, we present a retention time (tR) prediction model based on quantitative structure-retention relationship (QSRR). This model can predict the retention time of a given structure of phthalates including isomers. Twenty-three molecular descriptors were used in the development of the multivariate linear regression QSRR model. The regression coefficient (R2) between predicted and experimental retention times of 26 training set compounds was 0.9912. The combination of the retention time prediction model with identification via accurate mass search and target MS/MS spectrum interpretation can enhance the identification confidence in the lack of reference standards. Two previously unreported phthalate metabolites were identified in human urine, using this model. The results of this study showed that the developed QSRR model could be a useful tool to predict the retention times of unknown metabolites of phthalates and their alternatives in future non-targeted screening analysis. The concentration of these two unknown compounds was also estimated using a quantitative structure-ion intensity relationship (QSIIR) model.

Recent advances in non-targeted screening analysis using liquid chromatography - high resolution mass spectrometry to explore new biomarkers for human exposure.

Over the last decade, advances related to high-resolution mass spectrometry (HRMS) have led to improved capabilities for non-targeted chemical analyses. Important applications for these capabilities include identifying unknown xenobiotics and discovering emerging contaminants in human samples as exposure biomarkers. Despite technological advances, identifying unknown compounds by non-targeted analyses remains challenging due in part to the lack of MS spectral libraries and inherent sample complexity resulting in the generation of large amounts of MS data. While high resolution can separate nominally isobaric compounds in a mass spectrum, isomers cannot be distinguished. Much work also remains to develop models to predict both mass spectra and retention times for the unexplored regions of chemical space. In this review, we focus on recent advances and applications of non-targeted analyses using liquid chromatography - high-resolution mass spectrometry (LC-HRMS) in human biomonitoring, including sample preparation, molecular formula assignments, and prediction models for retention times and mass fragmentations, to enable and improve identifications of unknown chemicals. The purpose of this review is to improve our understanding of the applicability and limitations in both the analytical methods and data analysis aspects of non-targeted analysis in human exposure studies. We also discuss the challenges and prospects in this field for future research on sample preparation, identification confidence and accuracy, data processing tools, MS spectra comparability, liquid chromatographic retention time (RT) prediction algorithms, and quantitative capabilities.

Correlations of phthalate metabolites in urine samples from fertile and infertile men: Free-form concentration vs. conjugated-form concentration.

In previous studies, the total content of urinary phthalate metabolites was commonly used to evaluate human exposure to phthalates. However, phthalate metabolites are mainly present in urine in two forms, conjugated and free. These metabolite forms in urine are more relevant to the biotransformation pathways of the phthalates in humans. Therefore, the concentration of these forms is more relevant to exposure related health outcomes than total content. In this study, instead of measuring total content, the free- and conjugated-form concentrations of phthalate metabolites in the urine of fertile and infertile men were measured. The main metabolites in urine of both groups are monoethyl phthalate (MEP) and the di-(2-ethylhexyl) phthalate (DEHP) metabolites. The geometric means of their both conjugated- and free-forms in the infertile group were higher than in the fertile group, specifically, 24.3 and 43.4 µg/g creatinine vs 8.5 and 28.9 µg/g creatinine, respectively, for MEP, and 50.0 and 9.1 µg/g creatinine vs 39.1 and 8.4 µg/g creatinine, respectively for total DEHP metabolites. We investigated the correlations of free- and conjugated-form phthalate metabolite concentrations between the infertile and fertile group as well as among different phthalate metabolites. The results showed that there was a statistically significant difference between the infertile and fertile group for monobenzyl phthalate (MBzP) in both free-form and conjugated-form. However, there was only a statistically significant difference between the two groups for conjugated-form MEP and MEHP, and no statistically significant difference between the two groups for free-form MEP and MEHP. The results of the Pearson correlation test revealed that the correlations between DEHP metabolites and the correlations between mid-sized phthalate metabolites (mono-n-butyl phthalate (MnBP), mono-isobutyl phthalate (MiBP) and mono-benzyl phthalate (MBzP)) were stronger than between these two clusters of metabolites. This study is the first attempt to examine possible effects of conjugated-form concentrations of phthalate metabolites on human fertility. The results of this study suggest that conjugated-form and free-form concentrations of urinary phthalate metabolites may be appropriate biomarkers for assessing human exposure to phthalates and association with health outcomes.

Chromatographic method for quick estimation of DNA interaction potency of environmental pollutants.

The DNA interaction potency of a chemical has been defined in the present study as the degree of a chemical's ability to interact with DNA. An estimation method of such a potency has been established based on the peak reduction of an oligonucleotide probe resulting from its interaction with chemicals based on high-performance liquid chromatography. A DNA interaction potency equivalency (PEQ) also has been proposed to evaluate the relative interaction potency of test chemicals against benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE). Five known direct DNA interaction chemicals were employed to demonstrate the method. Two known inactive chemicals were used as negative controls. Both the potency and PEQ(50) values (PEQ of testing chemical at 50% of the probe peak reduction) of these five chemicals were determined as BPDE > phenyl glycidyl ether (PGE) > tetrachlorohydroquinone (Cl4HQ) > methyl methanesulfonate (MMS) > styrene-7,8-oxide (SO). Among the reactive chemicals, MMS was found to break the oligonucleotide into smaller fragments, whereas BPDE, PGE, and SO form covalent adducts with the oligonucleotide. In the latter case, the formation of multi-chemical-oligonucleotide adducts also was observed by mass spectrometry. The method was employed to estimate the DNA interaction potency equivalency of diesel vehicle exhaust gas to demonstrate the applicability of this approach in evaluating the interaction potency of environmental pollutants in both gas and liquid phases.

Effects of mixtures of polychlorinated biphenyls, methylmercury, and organochlorine pesticides on hepatic DNA methylation in prepubertal female Sprague-Dawley rats.

DNA methylation is one of the epigenetic mechanisms that regulates gene expression, chromosome structure, and stability. Our objective was to determine whether the DNA methylation system could be a target following in utero and postnatal exposure to human blood contaminants. Pregnant rats were dosed daily from gestation day 1 until postnatal day 21 with 2 dose levels of either organochlorine pesticides (OCP; 0.019 or 1.9 mg/kg/day), methylmercury chloride (MeHg; 0.02 or 2 mg/kg/day), polychlorinated biphenyls (PCBs; 0.011 or 1.1 mg/kg/day), or a mixture (Mix; 0.05, or 5 mg/kg/day) including all 3 groups of chemicals. Livers from 1 female offspring per litter were collected at postnatal day 29. Hepatic analysis revealed that the mRNA abundance for DNA methyltransferase (DNMT)-1, -3a, and -3b were significantly reduced by the high dose of PCB, that the high dose of MeHg also reduced mRNA levels for DNMT-1, and -3b, but that OCP had no significant effects compared with control. The high dose of PCB and Mix reduced the abundance of the universal methyl donor S-adenosylmethionine, and Mix also reduced global genome DNA methylation (5-methyl-deoxycytidine/5-methyl-deoxycytidine + deoxycytidine). The latter is consistent with pyrosequencing methylation analysis, revealing that the high-dose groups (except OCP) generally decreased the methylation of CpG sites (position -63 to -29) in the promoter of the tumor suppressor gene p16(INK4a). Overall, these hepatic results suggest that the DNA methylation system can be affected by exposure to high doses of blood contaminants, and that OCP is the least potent chemical group from the investigated mixtures.