Mass Spectral Library for DNA Adductomics.

Endogenous electrophiles, ionizing and non-ionizing radiation, and hazardous chemicals present in the environment and diet can damage DNA by forming covalent adducts. DNA adducts can form in critical cancer driver genes and, if not repaired, may induce mutations during cell division, potentially leading to the onset of cancer. The detection and quantification of specific DNA adducts are some of the first steps in studying their role in carcinogenesis, the physiological conditions that lead to their production, and the risk assessment of exposure to specific genotoxic chemicals. Hundreds of different DNA adducts have been reported in the literature, and there is a critical need to establish a DNA adduct mass spectral database to facilitate the detection of previously observed DNA adducts and characterize newly discovered DNA adducts. We have collected synthetic DNA adduct standards from the research community, acquired MSn (n = 2, 3) fragmentation spectra using Orbitrap and Quadrupole-Time-of-Flight (Q-TOF) MS instrumentation, processed the spectral data and incorporated it into the MassBank of North America (MoNA) database, and created a DNA adduct portal Web site (https://sites.google.com/umn.edu/dnaadductportal) to serve as a central location for the DNA adduct mass spectra and metadata, including the spectral database downloadable in different formats. This spectral library should prove to be a valuable resource for the DNA adductomics community, accelerating research and improving our understanding of the role of DNA adducts in disease.

Development and validation of a method for analysis of 25 cannabinoids in oral fluid and exhaled breath condensate.

Currently, there is a significant demand in forensic toxicology for biomarkers of cannabis exposure that, unlike ∆9-tetrahydrocannabinol, can reliably indicate time and frequency of use, be sampled with relative ease, and correlate with impairment. Oral fluid (OF) and exhaled breath condensate (EBC) are alternative, non-invasive sample matrices that hold promise for identifying cannabis exposure biomarkers. OF, produced by salivary glands, is increasingly utilized in drug screening due to its non-invasive collection and is being explored as an alternative matrix for cannabinoid analysis. EBC is an aqueous specimen consisting of condensed water vapor containing water-soluble volatile and non-volatile components present in exhaled breath. Despite potential advantages, there are no reports on the use of EBC for cannabinoid detection. This study developed a supported liquid extraction approach and LC-QqQ-MS dMRM analytical method for quantification of 25 major and minor cannabinoids and metabolites in OF and EBC. The method was validated according to the ANSI/ASB 036 standard and other published guidelines. LOQ ranged from 0.5 to 6.0 ng/mL for all cannabinoids in both matrices. Recoveries for most analytes were 60-90%, with generally higher values for EBC compared to OF. Matrix effects were observed with some cannabinoids, with effects mitigated by use of matrix-matched calibration. Bias and precision were within ± 25%. Method applicability was demonstrated by analyzing ten authentic OF and EBC samples, with positive detections of multiple analytes in both matrices. The method will facilitate comprehensive analysis of cannabinoids in non-invasive sample matrices for the development of reliable cannabis exposure biomarkers.

DNA Aptamer-Cyanine Complexes as Generic Colorimetric Small-Molecule Sensors.

Aptamers are promising biorecognition elements for sensors. However, aptamer-based assays often lack the requisite levels of sensitivity and/or selectivity because they typically employ structure-switching aptamers with attenuated affinity and/or utilize reporters that require aptamer labeling or which are susceptible to false positives. Dye-displacement assays offer a label-free, sensitive means for overcoming these issues, wherein target binding liberates a dye that is complexed with the aptamer, producing an optical readout. However, broad utilization of these assays has been limited. Here, we demonstrate a rational approach to develop colorimetric cyanine dye-displacement assays that can be broadly applied to DNA aptamers regardless of their structure, sequence, affinity, or the physicochemical properties of their targets. Our approach should accelerate the development of mix-and-measure assays that could be applied for diverse analytical applications.

Development of a DNA Adductome Mass Spectral Database.

Mass spectrometry-based DNA adductomics is an emerging approach for the human biomonitoring of hazardous chemicals. A mass spectral database of DNA adducts will be created for the scientific community to investigate the associations between chemical exposures, DNA damage, and disease risk.

Comprehensive analysis of synthetic cannabinoids and metabolites in oral fluid by online solid-phase extraction coupled to liquid chromatography-triple quadrupole-mass spectrometry.

The class of novel psychoactive substances known as synthetic cannabinoids (SC) includes illicit compounds that are sprayed on plant material and smoked or sold as liquids to be vaporized in e-cigarettes. In toxicological analysis of SC, fast analytical methods are needed for the detection and confirmation of parent drugs and metabolites at very low levels. While various analytical methods have been developed for SC in blood and urine, few are available for alternative matrices such as oral fluid (OF). There are numerous advantages to using OF as a sample matrix for SC analysis, including non-invasive collection, lesser risk of adulteration, and presence of both parent drug and metabolites. Here we report a validated online solid-phase extraction (online SPE) method coupled to LC-QqQ-MS for rapid confirmation and quantitation of 72 structurally diverse SC parent drugs and metabolites in OF with 2.5 min of preconcentration time and a total elution time of < 10 min. The use of online SPE for sample pretreatment facilitates rapid and consistent processing and greatly increases sample throughput. The method was fully validated according to relevant guidelines (ANSI/ASB Standard 036). Bias and precision values were within ± 20% for all compounds in human OF matrix. Method detection and quantitation limits ranged from 0.4 to 3.8 ng/mL and from 1.1 to 11.6 ng/mL, respectively. Recovery, matrix effects, process efficiency, carryover, and stability were also within acceptable limits for the majority of compounds. Successful application of the method was demonstrated using blank human OF fortified with SC in addition to a set of authentic OF specimens previously tested by another laboratory. Graphical abstract.

LC-MS/MS based detection and characterization of covalent glutathione modifications formed by reactive drug of abuse metabolites.

Conjugation with the tripeptide glutathione (GSH) is a common mechanism of detoxification of many endogenous and exogenous compounds. This phenomenon typically occurs through the formation of a covalent bond between the nucleophilic free thiol moiety of GSH and an electrophilic site on the compound of interest. While GSH adducts have been identified for many licit drugs, there is a lack of information on the ability of drugs of abuse to adduct GSH. The present study utilized a metabolic assay with GSH as a nucleophilic trapping agent to bind reactive drug metabolites formed in situ. Extracted ion MS spectra were collected via LC-QqQ-MS/MS for all potentially significant ions and examined for fragmentation common to GSH-containing compounds, followed by confirmation of adduction and structural characterization performed by LC-QTOF-MS/MS. In addition to the two positive controls, of the 14 drugs of abuse tested, 10 exhibited GSH adduction, with several forming multiple adducts, resulting in a total of 22 individual identified adducts. A number of these are previously unreported in the literature, including those for diazepam, naltrexone, oxycodone and Δ9-THC.

Changes in persistent organic pollutant levels from adolescence to young adulthood.

Elimination rates and their corresponding half-lives are conceptually important and intuitively accessible pharmacokinetic measures of toxicant elimination, but regression-based estimates are biased proportional to the degree of continuing (background) exposure. We propose an alternative estimator, the censored normal regression model, which uses all observations, but treats individuals whose initial level failed to exceed their follow-up level as censored observations to weight the regression estimates from those that declined between blood draws. In this manner, we derive the intrinsic elimination rate, the elimination rate free from ongoing exposure, as a parameter in a regression with an unobserved, latent dependent variable. We utilize sequential measurements of persistent organic pollutants (POPs) levels from adolescence to adulthood, a period of intense change in size and body composition, to quantify individual-level change within a community exposed to significant quantities of contaminants over an extended period of time. Although much research has been conducted on effects of POPs, far less attention has been given to vectors of intake and changes in toxicant levels during the life course. We apply exploratory factor analysis (EFA) to types and timing of consumption, along with physical behavioral characteristics, to identify a structure of seven underlying factors. Although several variables show factorial complexity, the latent constructs included an age/maturation and period-related factor, a nutritional composite, consumption prior to pregnancy, fish and fowl consumed during pregnancy, factors distinguishing body mass and weight from height, and bottom-feeding fish consumption. Unadjusted and adjusted half-lives using the censored normal regression estimator, as well as estimated half-lives from conventional log concentration regressions, are reported for PCB groupings, specific congeners, p,p'-DDE, and HCB.

Covalent thiol adducts arising from reactive intermediates of cocaine biotransformation.

Exposure to cocaine results in the depletion of hepatocellular glutathione and macromolecular protein binding in humans. Such cocaine-induced responses have generally been attributed to oxidative stress and reactive metabolites resulting from oxidative activation of the cocaine tropane nitrogen. However, little conclusive data exists on the mechanistic pathways leading to protein modification or the structure and specificity of cocaine-derived adduction products. We now report a previously uncharacterized route of cocaine bioactivation leading to the covalent adduction of biological thiols, including cysteine and glutathione. Incubation of cocaine with biological nucleophiles in an in vitro biotransformation system containing human liver microsomes identified a monooxygenase-mediated event leading to the oxidation of, and subsequent sulfhydryl addition to, the cocaine aryl moiety. Adduct structures were confirmed using ultra-high performance liquid chromatography coupled to high resolution, high mass accuracy mass spectrometry. Examination of assays containing transgenic bactosomes expressing single human cytochrome P450 isoforms determined the role of P450s 1A2, 2C19, and 2D6 in the oxidation process resulting in adduct formation. P450-catalyzed aryl epoxide formation and subsequent attack by free nucleophilic moieties is consistent with the resulting adduct structures, mechanisms of formation, and the empirical observation of multiple structural and stereo isomers. Analogous adduction mechanisms were maintained across all sulfhydryl-containing nucleophile models examined; N-acetylcysteine, glutathione, and a synthetic cysteine-containing hexapeptide. Predictive in silico calculations of molecular reactivity and electrophilicity/nucleophilicity were compared to the results of in vitro assay incubations in order to better understand the adduction process using the principles of hard and soft acid and base (HSAB) theory. This study elucidated a novel metabolic pathway that may be of particular significance to the clinical and forensic toxicology of cocaine and provides analytical tools and methods that can be applied to the determination of these conjugates in humans, opening a new area of research on cocaine biotransformation and toxicology.

Covalent adduction of nitrogen mustards to model protein nucleophiles.

Protein adducts have the potential to serve as unique biomarkers of exposure to compounds of interest. Many xenobiotics (or their metabolites) are electrophilic and therefore reactive with nucleophilic amino acid residues on proteins. Nitrogen mustards are reactive xenobiotics with potential use as chemical warfare agents (CWA) or agents of terrorist attack, in addition to being employed as chemotherapeutic agents. The present study utilized cysteine-, lysine-, and histidine-containing model peptides to characterize in vitro adduction of the nitrogen mustards mechloroethamine (HN-2) and tris-(2-chlorethyl)amine (HN-3) to these nucleophilic amino acid residues by means of liquid chromatography-tandem mass spectrometry. The study assessed the structure of adducts formed, the time course of adduct formation, concentration-response relationships, and temporal stability of adducts. Adduction was hypothesized to occur on all three model peptides via initial formation of a reactive aziridinium intermediate for both mechloroethamine and tris-(2-chlorethyl)amine, followed by covalent adduction to nucleophilic residues. While adduction was found to occur most readily with cysteine, it was also observed at lysine and histidine, demonstrating that adduction by mechloroethamine and tris-(2-chlorethyl)amine is possible at multiple nucleophilic sites. Following solid phase extraction cleanup, adducts formed with mechloroethamine were stable for up to three weeks. Adducts formed with tris-(2-chlorethyl)amine were less stable; however, hydrolyzed secondary adducts were observed throughout the three week period. This study demonstrates that the nitrogen mustards mechloroethamine and tris-(2-chlorethyl)amine form stable adducts with reactive protein nucleophiles other than cysteine.

Determination of 32 cathinone derivatives and other designer drugs in serum by comprehensive LC-QQQ-MS/MS analysis.

Recently, clandestine drug lab operators have attempted to bypass controlled substances laws and regulations with "designer" compounds chemically and pharmacologically similar to controlled substances. For example, "bath salts" have erupted onto the scene as "legal highs" containing cathinone analogs that have produced severe side effects in users worldwide. These products have sparked concern among law enforcement agencies, and emergency bans have been placed on the sale of such items. Despite the increasing number of designer drugs available, there are few comprehensive screening techniques for their detection and quantification in biological specimens. The liquid chromatography triple quadrupole tandem mass spectrometry (LC-QQQ-MS/MS) method presented here encompasses over thirty important compounds within the phenethylamine, tryptamine, and piperazine designer drug classes. Analytes were determined by LC-QQQ-MS/MS in the multiple-reaction monitoring mode after mixed-mode solid-phase extraction. The bioanalytical method was fully validated according to recommended international guidelines. The assay was selective for all analytes with acceptable accuracy and precision. Limits of quantification were in the range of 1-10 ng/mL for each compound with limits of detection near 10 pg/mL. In order to evaluate its applicability in a forensic toxicological setting, the validated method was used to analyze post-mortem specimens from two cases that were suspected of containing designer drugs. The method was able to identify and quantify seven of these compounds at concentrations as low as 11 ng/mL. The method should have wide applicability for rapid screening of important new drugs of abuse at high sensitivity in both post- and ante-mortem forensic analysis.

Evaluation of in vitro metabolic systems for common drugs of abuse. 1. Cocaine.

This study examined the efficacy of four common in vitro assay systems in producing metabolic profiles consistent with in vivo data for drugs of abuse. Cocaine (COC) was selected for this study because of its complex biotransformation pathways, diverse metabolic processes and because extensive Phase I and Phase II metabolomic examination of COC has not yet been reported by means of in vitro assay. COC metabolism was assessed with a series of common in vitro assay systems (human liver microsomes, cytosol and human liver S9 fraction and horseradish peroxidase) using liquid chromatography-tandem mass spectrometry with multiple reaction monitoring. Qualitative and quantitative differences in analyte production were noted among the various active Phase I and Phase II metabolic systems. Assay incubation time was found to be a determining factor in metabolic profile, specifically with primary versus secondary metabolite formation. Regioselective arene hydroxylation of COC was conclusively documented in human hepatic metabolic models, while peroxidase-based assay systems displayed less selectivity in oxidative aryl biotransformation. Results demonstrate the applicability of in vitro systems in studying COC metabolite production and the impact of assay selection and variation in method parameters on metabolite profiles for this important drug of abuse.

A culturally appropriate method for validating self-reported drug administration among indigenous people who use injection drugs.

Compared with other racial/ethnic groups in the United States (US), American Indians/Alaska Natives have one of the fastest climbing rates of drug overdose deaths involving stimulants. Validating the substances self-reported by Indigenous people who use injection drugs (IPWIDs) can present logistical and cultural challenges. While the collection of biospecimens (e.g., urine, blood, hair follicle) can be one way to cross-validate the substances self-reported by IPWIDs, the collection of biospecimens has been historically problematic when conducting substance use research with Indigenous North Americans. In our National Institutes of Health (NIH)-supported pilot research conducted with IPWIDs, we have documented low willingness to provide a biospecimen to a research team. This article demonstrates an alternative method for validating self-reported substances injected by IPWIDs that does not require the extraction of biospecimens from Indigenous bodies and spaces. The method described includes:•Collecting used, unwashed syringes from IPWIDs at the time of behavioral assessment,•Sampling the used syringe by washing the syringe needle/barrel with methanol,•Analyzing the samples with gas chromatography mass spectrometry (GC-MS) and liquid chromatography coupled to triple-quadrupole mass spectrometry (LC-QQQ-MS). This method offers a more culturally appropriate alternative to validate substances self-reported by IPWIDs during behavioral assessments.

Ferrate(VI) mediated degradation of the potent cyanotoxin, cylindrospermopsin: Kinetics, products, and toxicity.

The presence of cylindrospermopsin (CYN), a potent cyanotoxin, in drinking water sources poses a tremendous risk to humans and the environment. Detailed kinetic studies herein demonstrate ferrate(VI) (FeVIO42-, Fe(VI)) mediated oxidation of CYN and the model compound 6-hydroxymethyl uracil (6-HOMU) lead to their effective degradation under neutral and alkaline solution pH. A transformation product analysis indicated oxidation of the uracil ring, which has functionality critical to the toxicity of CYN. The oxidative cleavage of the C5=C6 double bond resulted in fragmentation of the uracil ring. Amide hydrolysis is a contributing pathway leading to the fragmentation of the uracil ring. Under extended treatment, hydrolysis, and extensive oxidation lead to complete destruction of the uracil ring skeleton, resulting in the generation of a variety of products including nontoxic cylindrospermopsic acid. The ELISA biological activity of the CYN product mixtures produced during Fe(VI) treatment parallels the concentration of CYN. These results suggest the products do not possess ELISA biological activity at the concentrations produced during treatment. The Fe(VI) mediated degradation was also effective in the presence of humic acid and unaffected by the presence of common inorganic ions under our experimental conditions. The Fe(VI) remediation of CYN and uracil based toxins appears a promising drinking water treatment process.

Evaluation of extraction parameters in authentic hair reference material using statistical design of experiments.

Optimal methods for forensic hair analysis are often debated, especially regarding extraction parameters that include incubation time, temperature, and size of extracted hair particles. To assess hair pretreatment parameters for analysis of drugs of abuse, the statistical technique known as Design of Experiments (DoE) is useful. DoE evaluates both the individual roles and the combinatorial associations between multiple variables and overall drug extraction efficiency. Previous reports have focused on incorporated hair reference material (HRM), which is prepared in the lab at a specified drug concentration. In contrast, authentic HRM, which is prepared by diluting hair from drug users with blank hair to achieve specific drug concentrations, is an effective matrix for standardization of forensic hair testing, since the drug is incorporated into the hair matrix via uptake from systemic distribution. In the present study, extraction parameters for authentic HRM samples containing multiple drugs (diazepam, alprazolam, cocaine, methamphetamine, oxycodone, hydrocodone, and morphine) and metabolites (nordiazepam, cocaethylene, norcocaine, hydroxycocaine, and 6-monoacetylmorphine) were optimized based on recovery using a 23 full factorial DoE matrix. The factors evaluated included extraction solvent volume/sample weight ratio (12.5 or 25 µL/mg), particle size (pulverized or cut into 1 mm snippets), and extraction time (2 or 24 h) using solvent swelling. DoE analysis revealed significant differences in the optimal combinations of extraction parameters for maximum recovery. However, for the majority of drugs and metabolites, the most effective extraction method consisted of pulverizing hair prior to a 2-h extraction with a 12.5 µL/mg extraction solvent volume/sample weight ratio.

Evaluation of Pretreatment and Extraction Parameters for the Analysis of Fentanyl in Hair Using Statistical Design of Experiments (DoE).

Optimal methods for hair analysis are often debated. Previous work in this laboratory demonstrated that the statistical technique known as Design of Experiments (DoE) is useful for such optimization. DoE evaluates both the individual roles and the combinatorial associations among multiple independent variables (i.e., hair pretreatment parameters) and a dependent variable (i.e., drug recovery from hair). In this study, hair externally contaminated with fentanyl underwent decontamination with combinations of parameters based on a 24 fractional factorial block design DoE matrix. The parameters of interest included aqueous wash solvent (1% sodium dodecyl sulfate (SDS) or water), organic wash solvent (dichloromethane or methanol), number of consecutive washes (one or three), sequence of washes (aqueous first or organic first) and wash time (30 s or 30 min). The optimal method for decontaminating fentanyl from the hair surface was found to be one 30-min wash with dichloromethane followed by one 30-min wash with water. Pretreatment parameters were optimized with a 23 full factorial DoE matrix using authentic hair reference material (HRM), which consisted of pooled drug user hair diluted to a known concentration of fentanyl with drug-free hair. The factors of interest were extraction solvent/sample weight ratio (12.5 or 25 µL/mg), hair particle size (pulverized or 1 mm segments) and extraction time (2 or 24 h). The most effective pretreatment method for fentanyl consisted of pulverizing the hair prior to a 2-h extraction in a 25 µL/mg extraction solvent volume/sample weight ratio. Finally, using the optimized pretreatment methods, fentanyl containing authentic HRM was extracted using aqueous base, solvent and enzymatic hair extraction methods, where it was determined that the aqueous base technique was most effective for recovery of fentanyl. These experiments further demonstrate the value of DoE and authentic HRM in method development for forensic hair analysis.

Quantitation and Validation of 34 Fentanyl Analogs from Liver Tissue Using a QuEChERS Extraction and LC-MS-MS Analysis.

Since 2013, drug overdose deaths involving synthetic opioids (including fentanyl and fentanyl analogs) have increased from 3,105 to 31,335 in 2018. Postmortem toxicological analysis in fentanyl-related overdose deaths is complicated by the high potency of the drug, often resulting in low analyte concentrations and associations with toxicity, multidrug use, novelty of emerging fentanyl analogs and postmortem redistribution. Objectives for this study include the development of a quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction and subsequent liquid chromatography-mass spectrometry--mass spectrometry analysis, validation of the method following the American Academy of Forensic Sciences Standards Board (ASB) standard 036 requirements and application to authentic liver specimens for 34 analytes including fentanyl, metabolites and fentanyl analogs. The bias for all 34 fentanyl analogs did not exceed ±10% for any of the low, medium or high concentrations and the %CV did not exceed 20%. No interferences were identified. All 34 analytes were within the criteria for acceptable percent ionization suppression or enhancement with the low concentration ranging from -10.2% to 23.7% and the high concentration ranging from -7.1% to 11.0%. Liver specimens from 22 authentic postmortem cases were extracted and analyzed with all samples being positive for at least one target analyte from the 34 compounds. Of the 22 samples, 17 contained fentanyl and metabolites plus at least one fentanyl analog. The highest concentration for a fentanyl analog was 541.4 µg/kg of para-fluoroisobutyryl fentanyl (FIBF). The concentrations for fentanyl (n = 20) ranged between 3.6 and 164.9 µg/kg with a mean of 54.7 µg/kg. The fentanyl analog that was most encountered was methoxyacetyl fentanyl (n = 11) with a range of 0.2-4.6 µg/kg and a mean of 1.3 µg/kg. The QuEChERS extraction was fully validated using the ASB Standard 036 requirements for fentanyl, metabolites and fentanyl analogs in liver tissue.

Tenuous dose-response correlations for common disease states: case study of cholesterol and perfluorooctanoate/sulfonate (PFOA/PFOS) in the C8 Health Project.

Persistent organic chemicals, such as perfluorooctanoic acid (PFOA), perfluorooctanesulfonate (PFOS), dioxins, and polychlorinated biphenyls, pose investigative challenges because they are found in virtually everyone (there is no unexposed control group). To overcome this problem, outcome data in some studies are sorted by chemical dose level and findings in low-end dose groups are compared to sequential higher dose groups. An example is the C8 Health Project that evaluated serum PFOA/PFOS (C8) and total cholesterol among 46,294 West Virginia residents who lived, worked, or went to school for at least 1 year in a C8 contaminated drinking-water district and were over age 18 in 2005-2006. The risk for high total cholesterol (>240 mg/dL) measured via odds ratios (ORs) in logistic regression models showed sequential OR increases with PFOA quartile, in comparison to the lowest quartile (OR = 1.00), that were each significantly elevated (OR = 1.21, 1.33, and 1.40, respectively), but age, sex, and body mass index were stronger correlates. Importantly, the magnitude of cholesterol increase was small (12 mg/dL from lowest to highest exposure deciles) and comparison to similar statistics for the general U.S. population showed the C8 cohort had lower rates of high cholesterol. This suggests that inadvertent selection bias may have affected the lowest exposure quartile (control group), making tenuous the dose-response relationship between PFOA/PFOS and risk of high cholesterol. This case illustrates the substantial difficulties in assigning toxicological importance to statistical comparisons for common disease states that utilize subgroups with low exposures as an effective control group.

Assessing Hair Decontamination Protocols for Diazepam, Heroin, Cocaine and Δ9-Tetrahydrocannabinol by Statistical Design of Experiments.

Prior to toxicological analysis, hair as a matrix requires pre-treatment measures including decontamination, homogenization and extraction. Decontamination is performed to differentiate between drug present from superficial deposition and drug incorporated from systemic distribution following ingestion. There are many methods for decontamination of hair samples, mostly developed by empirically using a traditional "one factor at a time" approach, in which one independent variable at a time is changed to observe the effect on the dependent variable. The goal of the present work was to compare the efficacy of decontamination protocols using statistical "design of experiments" (DoE), which allows for analysis of multiple variables and interactions within a single experiment. Decontamination parameters included identity of aqueous and organic wash solutions, number of sequential aqueous and organic washes, order of aqueous and organic washes, and duration of each wash. DoE studies were completed to identify optimal decontamination conditions for four abused drugs with varying physiochemical properties. For this purpose, drug-free human hair was externally contaminated with diazepam, heroin, cocaine or Δ9-tetrahydrocannabinol. Each analyte was found to have a unique set of decontamination conditions that were most effective. These included three 30-min washes with methanol followed by three with 1% sodium dodecyl sulfide for diazepam, three 30-s washes with dichloromethane followed by one with water for heroin, one 30-s wash with 1% sodium dodecyl sulfate followed by three with dichloromethane for cocaine and three 30-min washes with water followed by one with methanol for Δ9-tetrahydrocannabinol. The results provide proof-of-principle for a DoE approach to identify effective parameters for hair decontamination for a physicochemically diverse group of drugs. The major advantage of DoE is to elucidate combinations of parameters that result in effective removal of surface contamination, a goal that would be challenging to accomplish using a one factor at a time approach.

Persistent organic pollutants and anti-thyroid peroxidase levels in Akwesasne Mohawk young adults.

Persistent organic pollutants, such as polychlorinated biphenyls (PCBs), hexachlorobenzene (HCB), and p,p'-dichlorophenyldichloroethylene (DDE), have been found to elicit a broad spectrum of biologic, metabolic, and immunologic responses. The potential of these pollutants to impair immune responses and trigger autoimmune disease is of growing concern, given their structural similarity to thyroid hormones and their potential to modulate the mechanisms and interfere with the binding of these hormones. We examine the relationship of different groupings of PCBs, according to chlorination and structure, and of p,p'-DDE and HCB to anti-thyroid peroxidase antibody, a useful tool in the evaluation of thyroid dysfunction, among 115 young adults of the Akwesasne Mohawk Nation. Overall, 18 participants (15.4%) had anti-thyroid peroxidase antibodies (TPOAb) levels above the normal laboratory reference range (23% of females, 9% of males). Among participants who were breast fed (n=47), those with an elevated TPOAb level had significantly higher levels of all PCB groupings, with the exception of levels of non-persistent PCBs which did not differ significantly. Levels of p,p'-DDE were also significantly elevated, while HCB and mirex were not higher among those with elevated TPOAb. Also, after stratifying by breast-feeding status, participants who were breast fed showed significant, positive relationships between TPOAb levels and all PCB groupings, except groups comprised of non-persistent PCBs, and with p,p'-DDE, HCB, and mirex. No effects were evident among non-breast-fed young adults. Further studies are necessary to elucidate the site and mechanism of action of these persistent organic pollutants (POPs) and to establish thresholds for these effects, especially among populations with background levels of toxicant exposure.

Comparative covalent protein binding of 2,5-hexanedione and 3-acetyl-2,5-hexanedione in the rat.

2,5-Hexanedione (HD) is the metabolite implicated in n-hexane neurotoxicity. This gamma-diketone reacts with protein lysine amines to form 2,5-dimethylpyrrole adducts. Pyrrole adduction of neurofilaments (NF) and/or other axonal proteins was proposed as a critical step in the neuropathy. While pyrrole adduction is widely accepted as necessary, subsequent pyrrole oxidation, which may result in protein cross-linking, was alternatively postulated as the critical mechanistic step. Previous studies have indicated that 3-acetyl-2,5-HD (AcHD), an analogue that forms pyrroles that do not oxidize, was not neurotoxic in rats. However, relative levels of pyrrole adduction of NF or other axonal proteins were not reported. In the present study, groups of 6 male Wistar rats were given saline, [1,6-(14)C]-HD (3 mmol/kg/d), or [5-(14)C]-AcHD (0.1 mmol/kg/d), i.p. for 21 d. HD- and AcHD-treated rats lost 10% and gained 14% body weight, respectively, compared to a 22% gain for control rats. At termination, HD- and AcHD-treated rats exhibited mean scores of 3.5 and 1.4, respectively, for hindlimb weakness (0-5 scale). Incorporation of radiolabel from HD was 27.8 +/- 3.9, 13.9 +/- 2.6, and 7.8 +/- 0.6 nmol/mg in plasma protein, purified globin, and axonal cytoskeletal proteins, respectively, compared to 0.6 +/- 0.1, 1.6 +/- 0.5, and 1.0 +/- 0.1 for AcHD. Binding of HD to the NF-L, -M, and -H subunit proteins from treated animals was 4-, 24-, and 13-fold higher, respectively, that that of AcHD, indicating differing stoichiometry and patterns of NF adduction for the two diketones. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of globin and NF proteins did not demonstrate protein cross-linking for either diketone at the dose levels and time period examined. These results indicate that that the lack of neurotoxicity previously reported for AcHD may reflect differences in adduct levels at critical axonal target sites rather than an inability to form cross-linking adducts. Based on these data, further studies are required to fully assess the neurotoxic potency of AcHD and other non-cross-linking analogues as compared to HD.