Mona I. Churchwell

Placental transfer of the soy isoflavone genistein following dietary and gavage administration to Sprague Dawley rats

Genistein, the principal soy isoflavone, has estrogenic activity and is widely consumed by humans for putative beneficial health effects. The goal of the present study was to measure placental transfer of genistein in rats as a possible route of developmental exposure. Pregnant Sprague-Dawley rats were administered genistein orally, either by diet or by gavage. Concentrations of genistein aglycone and conjugates were measured in maternal and offspring serum and brain using HPLC with isotope dilution electrospray tandem mass spectrometry. Although fetal or neonatal serum concentrations of total genistein were approximately 20-fold lower than maternal serum concentrations, the biologically active genistein aglycone concentration was only 5-fold lower. Fetal brain contained predominately genistein aglycone at levels similar to those in the maternal brain. These studies show that genistein aglycone crosses the rat placenta and can reach fetal brain from maternal serum genistein levels that are relevant to those observed in humans.

Pharmacokinetics of bisphenol A in humans following a single oral administration.

BACKGROUND Human exposures to bisphenol A (BPA) are widespread. The current study addresses uncertainties regarding human pharmacokinetics of BPA. OBJECTIVE To reduce uncertainties about the metabolism and excretion of BPA in humans following oral administration. METHODS We exposed six men and eight women to 100 μg/kg bw of deuterated BPA (d6-BPA) by oral administration and conducted blood and urine analysis over a three day period. The use of d6-BPA allowed administered d6-BPA to be distinguished from background native (unlabeled) BPA. We calculated the rate of oral absorption, serum elimination, half-life, area under the curve (AUC), urinary excretion, and metabolism to glucuronide and sulfate conjugates. RESULTS Mean serum total (unconjugated and conjugated) d6-BPA Cmax of 1711 nM (390 ng/ml) was observed at Tmax of 1.1 ± 0.50h. Unconjugated d6-BPA appeared in serum within 5-20 min of dosing with a mean Cmax of 6.5 nM (1.5 ng/ml) observed at Tmax of 1.3 ± 0.52 h. Detectable blood levels of unconjugated or total d6-BPA were observed at 48 h in some subjects at concentrations near the LOD (0.001-0.002 ng/ml). The half-times for terminal elimination of total d6-BPA and unconjugated d6-BPA were 6.4 ± 2.0 h and 6.2 ± 2.6h, respectively. Recovery of total administered d6-BPA in urine was 84-109%. Most subjects (10 of 14) excreted >90% as metabolites within 24h. CONCLUSIONS Using more sensitive methods, our study expands the findings of other human oral pharmacokinetic studies. Conjugation reactions are rapid and nearly complete with unconjugated BPA comprising less than 1% of the total d6-BPA in blood at all times. Elimination of conjugates into urine largely occurs within 24h.

High-throughput quantification of soy isoflavones in human and rodent blood using liquid chromatography with electrospray mass spectrometry and tandem mass spectrometry detection

Soy-containing foods and dietary supplements are widely consumed for putative health benefits (e.g., cancer chemoprevention, beneficial effects on serum lipids associated with cardiovascular health, reduction of osteoporosis, relief of menopausal symptoms). However, studies of soy isoflavones in experimental animals suggest possible adverse effects as well (e.g., enhancement of reproductive organ cancer, modulation of endocrine function, anti-thyroid effects). This paper describes the development and validation of a sensitive high throughput method for quantifying isoflavones in blood from experimental animal and human studies. Serum samples containing genistein, daidzein, and equol were processed using reverse phase solid-phase extraction in the 96-well format for subsequent LC-ES/MS/MS or LC-ES/MS analysis using isotope dilution in conjunction with labeled internal standards. The method was validated by repetitive analysis of spiked blank serum and the intra-day and inter-day accuracy (88-99%) and precision (relative standard deviations from 3 to 13%) of measurement determined. The lower limit of quantification for all isoflavones was approximately 0.005 micro M using MS/MS detection, and 0.03 micro M using MS for genistein and daidzein. The degree of method performance, with respect to throughput, sensitivity and selectivity, makes this approach practical for analysis of large sample sets generated from mechanistic animal studies and human clinical trials of soy isoflavones.

Analysis of malachite green and metabolites in fish using liquid chromatography atmospheric pressure chemical ionization mass spectrometry

Malachite green (MG), a traditional agent used in aquaculture, is structurally related to other carcinogenic triphenylmethane dyes. Although MG is not approved for use in aquaculture, its low cost and high efficacy make illicit use likely. We developed sensitive and specific methods for determination of MG and its principal metabolite, leucoMG (LMG), in edible fish tissues using isotope dilution liquid chromatography atmosphere pressure chemical ionization mass spectrometry. MG and LMG concentrations were measured in filets from catfish treated with MG under putative use conditions (ca. 250 and 1000 ppb, respectively) and from commercial trout samples (0-3 and 0-96 ppb, respectively). Concentrations of LMG in edible fish tissues always exceeded those of MG. A rapid cone voltage switching acquisition procedure was used to simultaneously produce molecular ions for quantification and diagnostic fragment ions for confirmation of MG and metabolites. The accurate and precise agreement between diagnostic ion intensity ratios produced by LMG in authentic standards and incurred fish samples was used to unambiguously confirm the presence of LMG in edible fish tissue. This suggested the validity of using LMG as a marker residue for regulatory determination of MG misuse. Additional metabolites derived from oxidative metabolism of MG or LMG (demethylation and N-oxygenation) were identified in catfish and trout filets, including a primary arylamine which is structurally related to known carcinogens. The ability to simultaneously quantify residues of MG and LMG, and to confirm the chemical structure of a marker residue by using LC/MS, suggests that this procedure may be useful in monitoring the food supply for the unauthorized use of MG in aquaculture.

Identification of ceramides in human cells using liquid chromatography with detection by atmospheric pressure chemical ionization-mass spectrometry.

Ceramides are intermediates in the biosynthesis of membrane sphingolipids. These biomolecules are also important as second messengers in signal transduction pathways controlling cell growth. We have developed two reversed-phase high pressure liquid chromatography (RPHPLC) techniques for identification and quantification of ceramides from mammalian cells. One method was based on atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) detection of ceramides and had the advantage of requiring minimal sample preparation, yielding significant structural information, and affording high sensitivity. The second method relied on perbenzoylation of the ceramides and detection at 230 nm. The predominant ceramides detected in the human leukemic HL-60 cell were N-(palmitoyl)-sphingosine, N-(nervonyl)-sphingosine, and N-(lignoceroyl)-sphingosine. When selected ion monitoring was used with RPHPLC/APCI-MS, approximately 2.2 pmol N-(palmitoyl)-sphingosine and 1.7 pmol N-(nervonyl)-sphingosine were observed in an extract from 40,000 HL-60 cells. Perbenzoylation with benzoyl chloride permitted RPHPLC separation and 230 nm UV absorbance detection of the trisbenzoyl derivatives of sphingosine, N-(palmitoyl)-sphingosine, N-(nervonyl)-sphingosine, and N-(lignoceroyl)-sphingosine in the HL-60 cells. These results demonstrate the utility of utilizing two different methods coupled with APCI-MS for the quantification and identification of ceramides in biological samples.

Bisphenol A, Bisphenol S, and 4-Hydroxyphenyl 4-Isoprooxyphenylsulfone (BPSIP) in Urine and Blood of Cashiers

Background: Bisphenol A (BPA) is a high-production-volume chemical associated with a wide range of health outcomes in animal and human studies. BPA is used as a developer in thermal paper products, including cash register receipt paper; however, little is known about exposure of cashiers to BPA and alternative compounds in receipt paper. Objective: We determined whether handling receipt paper results in measurable absorption of BPA or the BPA alternatives bisphenol S (BPS) and 4-hydroxyphenyl 4-isoprooxyphenylsulfone (BPSIP). Methods: Cashiers (n = 77) and non-cashiers (n = 25) were recruited from the Raleigh–Durham–Chapel Hill region of North Carolina during 2011–2013. Receipts were analyzed for the presence of BPA or alternatives considered for use in thermal paper. In cashiers, total urine and serum BPA, BPS, and BPSIP levels in post-shift samples (collected ≤ 2 hr after completing a shift) were compared with pre-shift samples. Levels of these compounds in urine from cashiers were compared to levels in urine from non-cashiers. Results: Each receipt contained 1–2% by weight of the paper of BPA, BPS, or BPSIP. The post-shift geometric mean total urinary BPS concentration was significantly higher than the pre-shift mean in 33 cashiers who handled receipts containing BPS. The mean urine BPA concentrations in 31 cashiers who handled BPA receipts were as likely to decrease as to increase after a shift, but the mean post-shift concentrations were significantly higher than those in non-cashiers. BPSIP was detected more frequently in the urine of cashiers handling BPSIP receipts than in the urine of non-cashiers. Only a few cashiers had detectable levels of total BPA or BPS in serum, whereas BPSIP tended to be detected more frequently. Conclusions: Thermal receipt paper is a potential source of occupational exposure to BPA, BPS, and BPSIP. Citation: Thayer KA, Taylor KW, Garantziotis S, Schurman SH, Kissling GE, Hunt D, Herbert B, Church R, Jankowich R, Churchwell MI, Scheri RC, Birnbaum LS, Bucher JR. 2016. Bisphenol A, bisphenol S, and 4-hydroxyphenyl 4-isoprooxyphenylsulfone (BPSIP) in urine and blood of cashiers. Environ Health Perspect 124:437–444; http://dx.doi.org/10.1289/ehp.1409427

High-performance liquid chromatography—thermospray mass spectrometry of ten sulfonamide antibiotics: Analysis in milk at the ppb level

Ten sulfonamide antibiotics including sulfanilamide (SNL), sulfamethazine (SMZ), sulfamethizole (SMTZ), sulfachloropyridazine and sulfaquinoxaline (SQX), were analyzed by thermospray (TSP) mass spectrometry on-line with a high-performance liquid chromatography-UV detection system. Except for the pairs SMZ-SMTZ and sulfadimethoxine-SQX, the standards were resolved in both the UV and TSP profiles. Co-eluting compounds could be differentiated in TSP by their different relative molecular masses. The [M+H]+ ion was the base peak for all the standards except SNL, which showed an [M+NH4]+ ion. Collision-induced dissociation of the [M+H]+ ions afforded daughter ion spectra characterized by common ions at m/z 92, 108 and 156, and ions derived from the amine substituent ([MH-155]+). TSP detection limits [signal-to-noise ratio (S/N) > 3] were below 20 ng (scan mode), 2 ng (selected reaction monitoring, daughter ions from [M+H]+) and 400 pg (selected ion monitoring). UV detection limits were ca. 2 ng (S/N > 5). Results obtained from the multi-residue analysis of spiked cow milk samples at the low ng/ml level are presented.

Comparison of Life-Stage-Dependent Internal Dosimetry for Bisphenol A, Ethinyl Estradiol, a Reference Estrogen, and Endogenous Estradiol to Test an Estrogenic Mode of Action in Sprague Dawley Rats

Bisphenol A (BPA) was administered by gavage (2.5-300,000 μg/kg body weight (bw)/day) to pregnant Sprague Dawley dams, newborn pups, and continuing into adulthood. Aglycone (i.e., unconjugated and active) and conjugated (i.e., inactive) BPA were evaluated by liquid chromatography electrospray tandem mass spectrometry (LC-ES/MS/MS) in serum to better interpret toxicological endpoints measured in the study. Ethinyl estradiol (EE2, 0.5 and 5 μg/kg bw/day) and the endogenous hormones, 17β-estradiol (E2) and testosterone, were similarly evaluated. Mean BPA aglycone levels in vehicle and naïve control rat serum (0.02-0.5 ng/ml) indicated sample processing artifact, consistent with literature reports of a propensity for postexposure blood contamination by BPA. Direct measurements of BPA-glucuronide in vehicle and naïve control serum (2-10nM) indicated unintentional exposure and metabolism at levels similar to those produced by 2.5 μg/kg bw/day BPA (7-10nM), despite careful attention to potential BPA inputs (diet, drinking water, vehicle, cages, bedding, and dust) and rigorous dosing solution certification and delivery. The source of this exposure could not be identified, but interpretation of the toxicological effects, observed only at the highest BPA doses, was not compromised. Internal exposures to BPA and EE2 aglycones were highest in young rats. When maximal serum concentrations from the two highest BPA doses and both EE2 doses were compared with concurrent levels of endogenous E2, the ERα binding equivalents were similar to or above those of endogenous E2 in male and female rats of all ages tested. Such evaluations of estrogenic internal dosimetry and comprehensive evaluation of contamination impact should aid in extrapolating risks from human BPA exposures.

Photodecomposition of Pigment Yellow 74, a Pigment Used in Tattoo Inks¶

Tattooing has become a popular recreational practice among younger adults over the past decade. Although some of the pigments used in tattooing have been described, very little is known concerning the toxicology, phototoxicology or photochemistry of these pigments. Seven yellow tattoo inks were obtained from commercial sources and their pigments extracted, identified and quantitatively analyzed. The monoazo compound Pigment Yellow 74 (PY74; CI 11741) was found to be the major pigment in several of the tattoo inks. Solutions of commercial PY74 in tetrahydrofuran (THF) were deoxygenated using argon gas, and the photochemical reaction products were determined after exposure to simulated solar light generated by a filtered 6.5 kW xenon arc lamp. Spectrophotometric and high‐pressure liquid chromatography (HPLC) analyses indicated that PY74 photodecomposed to multiple products that were isolated using a combination of silica chromatography and reversed‐phase HPLC. Three of the major photodecomposition products were identified by nuclear magnetic resonance and mass spectrometry as N(2‐methoxyphenyl)‐3‐oxobutanamide (o‐acetoacetanisidide), 2‐(hydroxyimine)‐N‐(2‐methoxyphenyl)‐3‐oxobutanamide and N,N″‐bis(2‐methoxyphenyl)urea. These results demonstrate that PY74 is not photostable in THF and that photochemical lysis occurs at several sites in PY74 including the hydrazone and amide groups. The data also suggest that the use of PY74 in tattoo inks could potentially result in the formation of photolysis products, resulting in toxicity at the tattoo site after irradiation with sunlight or more intense light sources.

Quantification of deuterated bisphenol A in serum, tissues, and excreta from adult Sprague‐Dawley rats using liquid chromatography with tandem mass spectrometry

Bisphenol A (BPA) is an important industrial chemical used in the manufacture of polycarbonate plastic products, epoxy resin-based food can liners, and paper products. The presence of BPA in urine of >90% of Americans aged 6-60 suggests ubiquitous and frequent exposure and is problematic because of the potential for endocrine disruption. The ubiquity of environmental BPA in common laboratory supplies used for sample collection, storage, and analysis greatly increases the likelihood of false positive determinations, particularly at trace levels. The current study validated using liquid chromatography/tandem mass spectrometry (LC/MS/MS) in conjunction with deuterated BPA as the dosing material to circumvent contamination for high sensitivity quantifications in rat serum, tissues, urine, and feces. The methods described provided measurements of both estrogen receptor-active aglycone and metabolically deactivated conjugated forms of BPA, a distinction that is critical to assessing toxicological potential. The adequacy of the described methodology was substantiated by its utility in analyzing samples from rats treated orally with a 100 µg/kg body weight dose of d6-BPA. These results emphasize the challenges inherent in measuring BPA in biological samples and how employing stable isotope labeled dosing can facilitate pharmacokinetic studies needed to understand BPA metabolism and disposition. Such studies conducted in experimental animal models, in conjunction with properly validated human biomonitoring data, will be the basis for PBPK modeling of BPA in environmentally exposed humans.