Fetoplacental Disposition and Toxicity of Cadmium in Mice Lacking the Bcrp Transporter.

The environmental toxicant cadmium (Cd) impairs the growth of rodents and humans in utero which in turn heightens susceptibility to diseases later in life. We previously demonstrated that the maternal-facing efflux transporter, breast cancer resistance protein (human BCRP/ABCG2, mouse Bcrp/Abcg2) confers resistance against Cd toxicity in human trophoblasts. In the current study, we sought to determine whether the absence of Bcrp alters the fetoplacental disposition and toxicity of Cd in mice. Pregnant female wild-type (WT) and Bcrp-null mice (n = 9-10/group) were administered a single injection of saline (5 ml/kg) or CdCl2 (5 mg/kg) on gestational day (GD) 9. Following Cd treatment, Bcrp-null offspring were shorter and accumulated more Cd in their placentas on GD 17 compared to WT mice. Because Cd can adversely impact placentation and transplacental nutrient delivery in mice, multiple pathways were assessed using morphometrics and immunohistochemistry including placenta zonation, vasculature development, and nutrient transporter expression. Most notably, the placentas of Bcrp-null mice had reduced immunostaining of the cell adhesion marker, ß-catenin, and the trophoblast marker, cytokeratin, as well as decreased expression of divalent metal nutrient transporters (Dmt1, Zip14, and ZnT1) following Cd treatment. In summary, the absence of Bcrp expression increased placental concentrations of Cd which was associated with shorter fetal size that may be related to differential changes in molecular patterns of placental development and nutrition.

Placental BCRP transporter reduces cadmium accumulation and toxicity in immortalized human trophoblasts.

Cadmium (Cd) is a ubiquitous environmental metal detectable in most pregnant women. Animal and human studies demonstrate that in utero exposure to Cd reduces birth weight and impairs perinatal growth due to placental toxicity. BCRP is a prominent transporter that can efflux xenobiotics from the placenta. This study sought to investigate Cd transport and toxicity in cultured human BeWo trophoblasts with reduced expression and function of the placental barrier transporter BCRP. Knockdown (KD) of BCRP protein expression and function in BeWo trophoblasts increased the intracellular accumulation of Cd by 100% following treatment with 1 µM CdCl2. No change in the expression of Cd uptake transporters was observed between control and BCRP-KD cells. Reduced BCRP expression impaired viability of BeWo cells exposed to CdCl2 for 48 hr (BCRP-KD IC50: 11 µM, control cells IC50: 18 µM). Moreover, BCRP-KD cells were more sensitive to CdCl2-induced cytotoxicity compared to control BeWo cells. CdCl2 treatment strongly induced the expression of the metal-binding protein metallothionein (MT) in both control and BCRP-KD cells, with significantly greater MT upregulation in Cd-treated BCRP-KD cells. These data suggest that the BCRP transporter reduces Cd accumulation in syncytiotrophoblasts, which may be one mechanism to reduce subsequent toxicity to the placenta and developing fetus.

Prenatal Cadmium Exposure and Maternal Sex Steroid Hormone Concentrations across Pregnancy.

Cadmium exposure has been associated with adverse perinatal outcomes. One possible mechanism is endocrine disruption. Studies of non-pregnant adults suggest that cadmium impacts androgen production; here, we examined these associations during pregnancy. Participants in the Understanding Pregnancy Signals and Infant Development (UPSIDE) cohort provided biospecimens and questionnaire data in each trimester (n = 272). We quantified urinary cadmium, serum total testosterone (TT), estrone, estradiol, and estriol and serum free testosterone (fT). In adjusted longitudinal models, we examined sex steroid concentrations across pregnancy in relation to specific gravity-adjusted, ln-transformed cadmium concentrations. Additionally, we examined trimester-specific associations and stratified models by fetal sex. Results are presented as percent change (%∆) in hormone concentrations. In longitudinal models, higher cadmium concentrations were associated with lower fT across pregnancy (%∆ = -5.19, 95%CI: -8.33, -1.93), with no differences in other hormones observed. In trimester-specific models, higher cadmium concentrations were associated with lower TT in trimester 2 (%∆ = -15.26, 95%CI: -25.15, -4.06) and lower fT in trimester 3 (%∆ = -14.35, 95%CI: -19.75, -8.59). Associations with TT were stronger in pregnancies carrying female fetuses. Maternal cadmium exposure may be associated with reduced testosterone in pregnancy. Additional work is necessary to understand how alterations in gestational testosterone activity may impact pregnancy and child health.

Cadmium reduces growth of male fetuses by impairing development of the placental vasculature and reducing expression of nutrient transporters.

In utero exposure to the toxic metal cadmium (Cd) alters fetoplacental growth in rodents and has been inversely associated with birth weight and infant size in some birth cohorts. Moreover, studies suggest that Cd may have differential effects on growth and development according to offspring sex. The purpose of the current study was to evaluate changes in male and female fetoplacental development following a single injection of saline (5 ml/kg ip) or cadmium chloride (CdCl2, 2.5, 5 mg/kg, ip) on gestational day (GD) 9. By GD18, no changes in fetal or placental weights were observed after treatment with 2.5 mg/kg CdCl2. By comparison, the weight and length of male fetuses and their placentas were reduced following treatment with 5 mg/kg CdCl2 whereas no change was observed in females. In addition, the area of maternal and fetal blood vessels as well as the expression of the glucose transporters, Glut1 and Glut3, and the endothelial marker, CD34, were reduced in the placentas of CdCl2-treated male offspring compared to females. Interestingly, the placentas of females accumulated 80% more Cd than males after CdCl2 (5 mg/kg) administration. Female placentas also had higher concentrations of zinc and the zinc transporter Znt1 compared to males which may explain the limited changes in fetal growth observed following CdCl2 treatment. Taken together, disruption of vasculature development and reduced expression of glucose transporters in the placenta provide potential mechanisms underlying reduced fetal growth in male offspring despite the greater accumulation of Cd in female placentas.

Gadolinium during human pregnancy following administration of gadolinium chelate before pregnancy.

Gadolinium (Gd), a toxic rare earth element, has been shown to dissociate from chelating agents and bioaccumulate within tissues, raising concerns about the possibility of their remobilization during pregnancy with subsequent free Gd exposures to developing fetuses. Gd chelates are among the most commonly used magnetic resonance imaging (MRI) contrast agents. This investigation was undertaken after the detection of elevated Gd (800-1000× higher than the usual rare earth element levels) in preliminary unpublished studies from the placentae of subjects in the NIH ECHO/UPSIDE Rochester Cohort Study and unpublished studies from placentae analyzed in formalin-fixed placental specimens from Surgical Pathology at the University of Rochester. Fifteen pregnancies with elevated Gd were studied (12 first pregnancies and 3 second pregnancies). Maternal bloods were collected from all three trimesters, maternal, and cord (fetal) bloods at delivery as well as placental tissue. Breastmilk was also collected from selected mothers. It was determined that Gd was present in maternal bloods from all three trimesters, and in cord bloods and breastmilk in both first and second pregnancies. These results emphasize the need to fully appreciate the implications of pre-pregnancy exposure to Gd chelates and its potential effects on maternal and fetal health.

The lead and copper rule: Limitations and lessons learned from Newark, New Jersey.

Flint, Michigan reignited the public discourse surrounding lead contamination in drinking water with Newark, New Jersey recently experiencing its own lead-in-water crisis. Following Flint's experience, the Environmental Protection Agency proposed changes to the Lead and Copper Rule (LCR), but these changes may not produce better detection of contamination. LCR testing requirements were evaluated for their ability to predict or identify problems from the recent (2015-2019) Newark lead exceedance data. LCR compliance and water quality data were obtained from the New Jersey Department of Environmental Protection (NJDEP) website. Between 2002 and 2015, Newark sampled on a reduced sampling plan (50 samples once every 3 years), as required, for lead and copper. These samples were divided between Newark's two water sources with uneven sampling distribution across the city, further limiting the potential to identify a risk of lead in drinking water. Results suggest a more rigorous testing requirement may have identified the problem sooner. Limitations related to the LCR that prevented Newark water suppliers from earlier detection of lead risk will continue under the revised LCR. This article is categorized under:Engineering Water > Water, Health, and SanitationScience of Water > Water Quality.

Protective role of the placental efflux transporter BCRP/ABCG2 in the relationship between prenatal cadmium exposure, placenta weight, and size at birth.

BACKGROUND AND AIM: Placental efflux transporter proteins, such as BCRP, reduce the placental and fetal toxicity of environmental contaminants but have received little attention in perinatal environmental epidemiology. Here, we evaluate the potential protective role of BCRP following prenatal exposure to cadmium, a metal that preferentially accumulates in the placenta and adversely impacts fetal growth. We hypothesized that individuals with a reduced function polymorphism in ABCG2, the gene encoding BCRP, would be most vulnerable to the adverse impacts of prenatal cadmium exposure, notably, smaller placental and fetal size. METHODS: We measured cadmium in maternal urine samples at each trimester and in term placentas from UPSIDE-ECHO study participants (NY, USA; n = 269). We fit adjusted multivariable linear regression and generalized estimating equation models to examine log-transformed urinary and placental cadmium concentrations in relation to birthweight, birth length, placental weight, and fetoplacental weight ratio (FPR) and stratified models by ABCG2 Q141K (C421A) genotype. RESULTS: Overall 17% of participants expressed the reduced-function ABCG2 C421A variant (AA or AC). Placental cadmium concentrations were inversely associated with placental weight (ß = -19.55; 95%CI: -37.06, -2.04) and trended towards higher FPR (ß = 0.25; 95%CI: -0.01, 0.52) with stronger associations in 421A variant infants. Notably, higher placental cadmium concentrations in 421A variant infants were associated with reduced placental weight (ß = -49.42; 95%CI: 98.87, 0.03), and higher FPR (ß = 0.85, 95%CI: 0.18, 1.52), while higher urinary cadmium concentration was associated with longer birth length (ß = 0.98; 95%CI: 0.37, 1.59), lower ponderal index (ß = -0.09; 95%CI: 0.15, -0.03), and higher FPR (ß = 0.42; 95%CI: 0.14, 0.71). CONCLUSIONS: Infants with reduced function ABCG2 polymorphisms may be particularly vulnerable to the developmental toxicity of cadmium as well as other xenobiotics that are BCRP substrates. Additional work examining the influence of placental transporters in environmental epidemiology cohorts is warranted.

A surface sampling and liquid chromatography mass spectrometry method for the analysis of quaternary ammonium compounds collected from public transportation buses in New Jersey.

Quaternary ammonium compounds (QACs) are a class of antimicrobial disinfectants whose use in cleaning products increased during the COVID-19 pandemic. Chemically, their low vapor pressure indicates a proclivity to persist on surfaces, and their presence suggests a level of protection against microorganisms. The widespread application of QACs in response to the SARS CoV-2 virus created a need to evaluate their longevity on surfaces, for both efficacy and possible health risks. There are however, no standardized analytical methods for QAC surface sampling and analysis, and no published studies quantifying their concentrations on mass transportation vehicles-a high occupancy, close-contact microenvironment documented to facilitate the spread the SARS CoV-2 virus. Here, we describe a robust liquid chromatography mass spectrometry (LC-MS) method for the analysis of QACs and simultaneous development of a direct surface sampling and extraction protocol. We demonstrate the applicability of the method through the analysis of surface samples collected from in-service public transportation buses. The rapid, sensitive LC-MS method included 8 target QACs quantified on a Q-Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer using an electrospray ionization source and Dionex UltiMate 3000 UHPLC system for analyte separation. QAC standard mixtures at concentrations between 0.1 ng mL-1 and 2000 ng mL-1 were analyzed, and chromatographic separation of all analytes was achieved in less than 10 min. All correlation coefficients were reported at r > 0.986, and LODs ranged from 0.007 to 2.103 ng mL-1 for all compounds, confirming the method's sensitivity. A previously reported surface sampling and extraction protocol was modified to further simplify the procedure and expand the number of target compounds. The new sampling protocol was optimized from 10 commercially available wipes and 4 solvent types by quantifying recovery from the surface. Band-Aid brand small gauze pads saturated with isopropanol had the highest recovery efficiencies, ranging from 61.5 to 102.9% across all analytes. To test the real-world applicability, wipe samples were collected from 4 in-circulation New Jersey Transit buses on 5 separate days over the course of a month to assess the occurrence and longevity of QACs on sanitized mass transportation vehicles. Concentrations of QACs were detected on every wipe sample taken, and at all sampled time points, confirming their persistence on hard surfaces. QACs have the potential to form polymers, and detection of the polymer might serve as a secondary indication of their effectiveness on surfaces. None of the polymers detected however, were unique to QACs from this study. The polymers detected were already present in the wipe and used as an internal standard to demonstrate the efficacy of extraction and analysis of polymeric QACs.

Dose Uptake of Platinum- and Ruthenium-based Compound Exposure in Zebrafish by Inductively Coupled Plasma Mass Spectrometry with Broader Applications.

Metals and metal-based compounds comprise multifarious pharmaco-active and toxicological xenobiotics. From heavy metal toxicity to chemotherapeutics, the toxicokinetics of these compounds have both historical and modern-day relevance. Zebrafish have become an attractive model organism in elucidating pharmaco- and toxicokinetics in environmental exposure and clinical translation studies. Although zebrafish studies have the benefit of being higher-throughput than rodent models, there are several significant constraints to the model. One such limitation is inherent in the waterborne dosing regimen. Water concentrations from these studies cannot be extrapolated to provide reliable internal dosages. Direct measurements of the metal-based compounds allow for a better correlation with compound-related molecular and biological responses. To overcome this limitation for metals and metal-based compounds, a technique was developed to digest zebrafish larval tissue after exposure and quantify metal concentrations within tissue samples by inductively coupled plasma mass spectrometry (ICPMS). ICPMS methods were used to determine the metal concentrations of platinum (Pt) from cisplatin and ruthenium (Ru) from several novel Ru-based chemotherapeutics in zebrafish tissue. Additionally, this protocol distinguished concentrations of Pt that were sequestered in the chorion of the larval compared with the zebrafish tissue. These results indicate that this method can be applied to quantitate the metal dose present in larval tissues. Further, this method may be adjusted to identify specific metals or metal-based compounds in a broad range of exposure and dosing studies.

Moving toward a Handheld "Plasma" Spectrometer for Elemental Analysis, Putting the Power of the Atom (Ion) in the Palm of Your Hand.

Many of the current innovations in instrument design have been focused on making them smaller, more rugged, and eventually field transportable. The ultimate application is obvious, carrying the instrument to the field for real time sample analysis without the need for a support laboratory. Real time data are priceless when screening either biological or environmental samples, as mitigation strategies can be initiated immediately upon the discovery that contaminant metals are present in a location they were not intended to be. Additionally, smaller "handheld" instruments generally require less sample for analysis, possibly increasing sensitivity, another advantage to instrument miniaturization. While many other instruments can be made smaller just by using available micro-technologies (e.g., eNose), shrinking an ICP-MS or AES to something someone might carry in a backpack or pocket is now closer to reality than in the past, and can be traced to its origins based on a component-by-component evaluation. While the optical and mass spectrometers continue to shrink in size, the ion/excitation source remains a challenge as a tradeoff exists between excitation capabilities and the power requirements for the plasma's generation. Other supporting elements have only recently become small enough for transport. A systematic review of both where the plasma spectrometer started and the evolution of technologies currently available may provide the roadmap necessary to miniaturize the spectrometer. We identify criteria on a component-by-component basis that need to be addressed in designing a miniaturized device and recognize components (e.g., source) that probably require further optimization. For example, the excitation/ionization source must be energetic enough to take a metal from a solid state to its ionic state. Previously, a plasma required a radio frequency generator or high-power DC source, but excitation can now be accomplished with non-thermal (cold) plasma sources. Sample introduction, for solids, liquids, and gasses, presents challenges for all sources in a field instrument. Next, the interface between source and a mass detector usually requires pressure reduction techniques to get an ion from plasma to the spectrometer. Currently, plasma mass spectrometers are field ready but not necessarily handheld. Optical emission spectrometers are already capable of getting photons to the detector but could eventually be connected to your phone. Inert plasma gas generation is close to field ready if nitrogen generators can be miniaturized. Many of these components are already commercially available or at least have been reported in the literature. Comparisons to other "handheld" elemental analysis devices that employ XRF, LIBS, and electrochemical methods (and their limitations) demonstrate that a "cold" plasma-based spectrometer can be more than competitive. Migrating the cold plasma from an emission only source to a mass spectrometer source, would allow both analyte identification and potentially source apportionment through isotopic fingerprinting, and may be the last major hurdle to overcome. Finally, we offer a possible design to aid in making the cold plasma source more applicable to a field deployment.

BCRP/ABCG2 Transporter Regulates Accumulation of Cadmium in Kidney Cells: Role of the Q141K Variant in Modulating Nephrotoxicity.

Exposure to the environmental pollutant cadmium is ubiquitous, as it is present in cigarette smoke and the food supply. Over time, cadmium enters and accumulates in the kidneys, where it causes tubular injury. The breast cancer resistance protein (BCRP, ATP-Binding Cassette G2 ABCG2) is an efflux transporter that mediates the urinary secretion of pharmaceuticals and toxins. The ABCG2 genetic variant Q141K exhibits altered membrane trafficking that results in reduced efflux of BCRP substrates. Here, we sought to 1) evaluate the in vitro and in vivo ability of BCRP to transport cadmium and protect kidney cells from toxicity and 2) determine whether this protection is impaired by the Q141K variant. Cadmium concentrations, cellular stress, and toxicity were quantified in human embryonic kidney 293 cells expressing an empty vector (EV), BCRP wild-type (WT), or variant (Q141K) gene. Treatment with CdCl2 resulted in greater accumulation of cadmium and apoptosis in EV cells relative to WT cells. Exposure to CdCl2 induced expression of stress-related genes and proteins including MT-1A/MT-2A, NAD(P)H quinone dehydrogenase 1, and heme oxygenase-1 to a higher extent in EV cells compared with WT cells. Notably, the Q141K variant protected against CdCl2-induced activation of stress genes and cytotoxicity, but this protection was to a lesser magnitude than observed with WT BCRP. Lastly, concentrations of cadmium in the kidneys of Bcrp knockout mice were 40% higher than in WT mice, confirming that cadmium is an in vivo substrate of BCRP. In conclusion, BCRP prevents the accumulation of cadmium and protects against toxicity, a response that is impaired by the Q141K variant. SIGNIFICANCE STATEMENT: The breast cancer resistance protein transporter lowers cellular accumulation of the toxic heavy metal cadmium. This protective function is partially attenuated by the Q141K genetic variant in the ABCG2 gene.

Translating Analytical Techniques in Geochemistry to Environmental Health.

From human health exposure related to environmental contamination to ancient deep-Earth processes related to differentiation of the Earth's geochemical reservoirs, the adaptability of inductively coupled plasma mass spectrometry (ICP-MS) has proven to be an indispensable standard technique that transcends disciplines. Continued advancements in ICP-MS, including improved auxiliary applications such as laser ablation (LA), ion/liquid chromatography (IC), automated pre-concentration systems (e.g., seaFAST), and improved desolvating nebulizer systems (e.g., Aridus and Apex) have revolutionized our ability to analyze almost any sample matrix with remarkable precision at exceedingly low elemental abundances. The versatility in ICP-MS applications allows for effective interdisciplinary crossover, opening a world of analytical possibilities. In this communication, we discuss the adaptability of geochemical techniques, including sample preparation and analysis, to environmental and biological systems, using Pb isotopes for source apportionment as a primary example.

Ruthenium-Cyclopentadienyl Bipyridine-Biotin Based Compounds: Synthesis and Biological Effect.

Prospective anticancer metallodrugs should consider target-specific components in their design in order to overcome the limitations of the current chemotherapeutics. The inclusion of vitamins, which receptors are overexpressed in many cancer cell lines, has proven to be a valid strategy. Therefore, in this paper we report the synthesis and characterization of a set of new compounds [Ru(η5-C5H5)(P(C6H4R)3)(4,4'-R'-2,2'-bpy)]+ (R = F and R' = H, 3; R = F and R' = biotin, 4; R = OCH3 and R' = H, 5; R = OCH3 and R' = biotin, 6), inspired by the exceptional good results recently obtained for the analogue bearing a triphenylphosphane ligand. The precursors for these syntheses were also described following modified literature procedures, [Ru(η5-C5H5)(P(C6H4R)3)2Cl], where R is -F (1) or -OCH3 (2). The structure of all compounds is fully supported by spectroscopic and analytical techniques and by X-ray diffraction studies for compounds 2, 3, and 5. All cationic compounds are cytotoxic in the two breast cancer cell lines tested, MCF7 and MDA-MB-231, and much better than cisplatin under the same experimental conditions. The cytotoxicity of the biotinylated compounds seems to be related with the Ru uptake by the cells expressing biotin receptors, indicating a potential mediated uptake. Indeed, a biotin-avidin study confirmed that the attachment of biotin to the organometallic fragment still allows biotin recognition by the protein. Therefore, the biotinylated compounds might be potent anticancer drugs as they show cytotoxic effect in breast cancer cells at low dose dependent on the compounds' uptake, induce cell death by apoptosis and inhibit the colony formation of cancer cells causing also less severe side effects in zebrafish.

A novel screening method for transition metal-based anticancer compounds using zebrafish embryo-larval assay and inductively coupled plasma-mass spectrometry analysis.

As novel metallodrugs continue to emerge, they are evaluated using models, including zebrafish, that offer unique sublethal endpoints. Testing metal-based anticancer compounds with high-throughput zebrafish toxicological assays requires analytical methods with the sensitivity to detect these sublethal tissue doses in very small sample masses (e.g., egg mass 100 µg). A robust bioanalytical model, zebrafish embryos coupled with inductively coupled plasma-mass spectrometry (ICPMS) for measurement of delivered dose, creates a very effective means for screening metal-based chemotherapeutic agents. In this study, we used ICPMS quantitation with the zebrafish embryo assays to detect metal equivalents at multiple response endpoints for two compounds, the chemotherapeutic agent cisplatin and ruthenium (Ru)-based prospective metallodrug, PMC79. We hypothesized that cisplatin and PMC79 have different mechanisms for inducing apoptosis and result in similar lesions but different potencies following water-borne exposure. An ICPMS method was developed to detect the metal in waterborne solution and tissue (detection limit: 5 parts per trillion for Ru or platinum [Pt]). The Ru-based compound was more potent (LC50 : 7.8 µm) than cisplatin (LC50 : 158 µm) and induced disparate lesions. Lethality from cisplatin exposure exhibited a threshold (values >15 mg/L) while no threshold was observed for delayed hatching (lowest observed adverse effect level 3.75 mg/L cisplatin; 8.7 Pt (ng)/organism). The Ru organometallic did not have a threshold for lethality. Cisplatin-induced delayed hatching was investigated further by larval-Pt distribution and preferentially distributed to the chorion. We propose that zebrafish embryo-larval assays coupled with ICPMS serve as a powerful platform to evaluate relative potency and toxic effects of metallodrug candidates.

Unprecedented inhibition of P-gp activity by a novel ruthenium-cyclopentadienyl compound bearing a bipyridine-biotin ligand.

Two new ruthenium complexes, [Ru(η5-Cp)(PPh3)(2,2'-bipy-4,4'-R)]+ with R = -CH2OH (Ru1) or dibiotin ester (Ru2) were synthesized and fully characterized. Both compounds were tested against two types of breast cancer cells (MCF7 and MDA-MB-231), showing better cytotoxicity than cisplatin in the same experimental conditions. Since multidrug resistance (MDR) is one of the main problems in cancer chemotherapy, we have assessed the potential of these compounds to overcome resistance to treatments. Ru2 showed exceptional selectivity as P-gp inhibitor, while Ru1 is possibly a substrate. In vivo studies in zebrafish showed that Ru2 is well tolerated up to 1.17 mg/L, presenting a LC50 of 5.73 mg/L at 5 days post fertilization.