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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.

Over the last decade, significant progress been made in elucidating the role of membrane transporters in altering drug disposition, with important toxicological consequences due to changes in localized concentrations of compounds. The topic of "Transporters and Toxicity" was recently highlighted as a scientific session at the International Transporter Consortium (ITC) Workshop 4 in 2021. The current white paper is not intended to be an extensive review on the topic of transporters and toxicity but an opportunity to highlight aspects of the role of transporters in various toxicities with clinically relevant implications as covered during the session. This includes a review of the role of solute carrier transporters in anticancer drug-induced organ injury, transporters as key players in organ barrier function, and the role of transporters in metal/metalloid toxicity.

Bile acid metabolism is altered in neonates on parenteral nutrition (PN), predisposing them to parenteral nutrition-associated liver disease. Cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in the bile acid synthesis pathway, is repressed by fibroblast growth factor 19 (FGF19) and phytosterols (PS). We describe a case of a preterm infant who developed necrotizing enterocolitis (NEC) and received exclusive PN for over 2 months. Our objective was to serially assess CYP7A1 activity and plasma FGF19 and PS concentrations in this infant case compared to five healthy preterm infants. We found that CYP7A1 activity increased during the first 2 weeks of life in control infants but was undetectable in the infant case. FGF19 concentrations were high at birth in all infants and subsequently declined and did not differ between the case and control infants. As expected, PS concentrations were elevated in the infant case and continued to increase despite lipid minimization. In conclusion, CYP7A1 activity was gradually upregulated in healthy preterm infants but remained suppressed in the infant requiring prolonged PN. Preterm infants also had elevated FGF19 concentrations at birth, which decreased with advancing postnatal age.

Traditional experimental testing to identify endocrine disruptors that enhance estrogenic signaling relies on expensive and labor-intensive experiments. We sought to design a knowledge-based deep neural network (k-DNN) approach to reveal and organize public high-throughput screening data for compounds with nuclear estrogen receptor α and β (ERα and ERβ) binding potentials. The target activity was rodent uterotrophic bioactivity driven by ERα/ERβ activations. After training, the resultant network successfully inferred critical relationships among ERα/ERβ target bioassays, shown as weights of 6521 edges between 1071 neurons. The resultant network uses an adverse outcome pathway (AOP) framework to mimic the signaling pathway initiated by ERα and identify compounds that mimic endogenous estrogens (i.e., estrogen mimetics). The k-DNN can predict estrogen mimetics by activating neurons representing several events in the ERα/ERβ signaling pathway. Therefore, this virtual pathway model, starting from a compound’s chemistry initiating ERα activation and ending with rodent uterotrophic bioactivity, can efficiently and accurately prioritize new estrogen mimetics (AUC = 0.864–0.927). This k-DNN method is a potential universal computational toxicology strategy to utilize public high-throughput screening data to characterize hazards and prioritize potentially toxic compounds.

Migration and subsequent invasion of extravillous trophoblasts into the uterus is essential for proper formation of the placenta. Disruption of these processes may result in poor pregnancy outcomes including preeclampsia, placenta accreta, fetal growth restriction, or fetal death. Currently, there are several methods for quantifying cell migration and invasion in vitro, each with limitations. Therefore, we developed a novel, high-throughput method to screen chemicals for their ability to alter human trophoblast migration. Human HTR8/SVneo trophoblast cells were cultured in Oris™ cell migration plates containing stopper barriers. After EVT cells attached and chemicals were added to media, stoppers were removed thereby creating a cell-free detection zone for migration. Entry of trophoblasts into this zone was monitored through imaging every 6 h and used to calculate a relative cell density. Chemicals known to increase (epidermal growth factor) and decrease (pertussis toxin and cadmium) trophoblast migration were used to validate this in vitro method. Next, a panel of environmental chemicals including bisphenols, mycoestrogens, and flame retardants, were screened for their ability to alter trophoblast invasion. In conclusion, a real-time method to track extravillous trophoblast migration offers potential for screening contaminants as placental toxicants.

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 CdCl(2) resulted in greater accumulation of cadmium and apoptosis in EV cells relative to WT cells. Exposure to CdCl(2) 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 CdCl(2)-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.

As defined by the World Health Organization, an endocrine disruptor is an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, its progeny, or (sub)populations. Traditional experimental testing regimens to identify toxicants that induce endocrine disruption can be expensive and time-consuming. Computational modeling has emerged as a promising and cost-effective alternative method for screening and prioritizing potentially endocrine-active compounds. The efficient identification of suitable chemical descriptors and machine-learning algorithms, including deep learning, is a considerable challenge for computational toxicology studies. Here, we sought to apply classic machine-learning algorithms and deep-learning approaches to a panel of over 7500 compounds tested against 18 Toxicity Forecaster assays related to nuclear estrogen receptor (ERα and ERβ) activity. Three binary fingerprints (Extended Connectivity FingerPrints, Functional Connectivity FingerPrints, and Molecular ACCess System) were used as chemical descriptors in this study. Each descriptor was combined with four machine-learning and two deep- learning (normal and multitask neural networks) approaches to construct models for all 18 ER assays. The resulting model performance was evaluated using the area under the receiver- operating curve (AUC) values obtained from a fivefold cross-validation procedure. The results showed that individual models have AUC values that range from 0.56 to 0.86. External validation was conducted using two additional sets of compounds (n = 592 and n = 966) with established interactions with nuclear ER demonstrated through experimentation. An agonist, antagonist, or binding score was determined for each compound by averaging its predicted probabilities in relevant assay models as an external validation, yielding AUC values ranging from 0.63 to 0.91. The results suggest that multitask neural networks offer advantages when modeling mechanistically related endpoints. Consensus predictions based on the average values of individual models remain the best modeling strategy for computational toxicity evaluations. Computational modeling has emerged as a promising and cost-effective alternative method for screening potentially endocrine active compounds. This study applies classic machine learning algorithms and deep learning approaches to a panel of over 7500 compounds tested against 18 Toxicity Forecaster assays related to nuclear estrogen receptor (ERα and ERβ) activity.

Low oxygen concentration, or hypoxia, is an important physiological regulator of placental function including chemical disposition. Here, we compared the ability of low oxygen tension to alter the expression of solute carriers (SLC) and ABC transporters in two human placental models, namely BeWo cells and term placental explants. We found that exposure to low oxygen concentration differentially regulates transporter expression in BeWo cells, including downregulation of ENT1, OATP4A1, OCTN2, BCRP, and MRP2/3/5, and upregulation of CNT1, OAT4, OATP2B1, SERT, SOAT, and MRP1. Similar upregulation of MRP1 and downregulation of MRP5 and BCRP were observed in explants, whereas uptake transporters were decreased or unchanged. Furthermore, a screening of transcriptional regulators of transporters revealed that hypoxia leads to a decrease in the mRNA levels of aryl hydrocarbon receptor, nuclear factor erythroid 2-related factor 2, and retinoid x receptor alpha in both human placental models. These data suggest that transporter expression is differentially regulated by oxygen concentration across experimental human placental models.

Introduction: The breast cancer resistance protein (BCRP/ABCG2) is a member of the ATP-binding cassette superfamily of transporters. Using the energy garnered from the hydrolysis of ATP, BCRP actively removes drugs and endogenous molecules from the cell. With broad expression across the liver, kidney, brain, placenta, testes, and small intestines, BCRP can impact the pharmacokinetics and pharmacodynamics of xenobiotics.Areas covered: The purpose of this review is to summarize the transcriptional signaling pathways that regulate BCRP expression across various tissues and mammalian species. We will cover the endobiotic- and xenobiotic-activated transcription factors that regulate the expression and activity of BCRP. These include the estrogen receptor, progesterone receptor, peroxisome proliferator-activated receptor, constitutive androstane receptor, pregnane X receptor, nuclear factor e2-related factor 2, and aryl hydrocarbon receptor.Expert opinion: Key transcription factors regulate BCRP expression and function in response to hormones and xenobiotics. Understanding this regulation provides an opportunity to improve pharmacotherapeutic outcomes by enhancing the efficacy and reducing the toxicity of drugs that are substrates of this efflux transporter.

Hepatotoxicity is a leading cause of attrition in the drug development process. Traditional preclinical and clinical studies to evaluate hepatotoxicity liabilities are expensive and time consuming. With the advent of critical advancements in high-throughput screening, there has been a rapid accumulation of in vitro toxicity data available to inform the risk assessment of new pharmaceuticals and chemicals. To this end, we curated and merged all available in vivo hepatotoxicity data obtained from the literature and public resources, which yielded a comprehensive database of 4089 compounds that includes hepatotoxicity classifications. After dividing the original database of chemicals into modeling and test sets, PubChem assay data were automatically extracted using an in-house data mining tool and clustered based on relationships between structural fragments and cellular responses in in vitro assays. The resultant PubChem assay clusters were further investigated. During the cross-validation procedure, the biological data obtained from several assay clusters exhibited high predictivity of hepatotoxicity and these assays were selected to evaluate the test set compounds. The read-across results indicated that if a new compound contained specific identified chemical fragments (ie, Molecular Initiating Event) and showed active responses in the relevant selected PubChem assays, there was potential for the chemical to be hepatotoxic in vivo. Furthermore, several mechanisms that might contribute to toxicity were derived from the modeling results including alterations in nuclear receptor signaling and inhibition of DNA repair. This modeling strategy can be further applied to the investigation of other complex chemical toxicity phenomena (eg, developmental and reproductive toxicities) as well as drug efficacy.

Since the discovery of calbindin release into the urine during renal injury, there has been growing interest in the utility of this protein as a biomarker of nephrotoxicity. However, little is known about the intrarenal regulation of the release and expression of this calcium-regulating protein during kidney injury. We sought to characterize the time-dependent expression and excretion of the protein calbindin in the distal tubule in comparison to kidney injury molecule-1 (Kim-1), a protein in the proximal tubule, in mice treated with cisplatin. Urine, blood, and kidneys were collected from male C57BL/6 mice treated with vehicle or cisplatin (20 mg/kg ip). Urinary concentrations of calbindin and Kim-1 were elevated by 11.6-fold and 2.5-fold, respectively, within 2 days after cisplatin. Circulating creatinine and blood urea nitrogen levels increased in cisplatin-treated mice by 3 days, confirming the development of acute kidney injury. Time-dependent decreases in intrarenal calbindin protein were observed on Days 3 and 4 and a 200-fold upregulation of calbindin (CALB1) and KIM-1 messenger RNAs (mRNAs) was observed on Day 3. These data suggest that early loss of calbindin protein into the urine along with declines in renal calbindin levels initiates a compensatory induction of mRNA expression at later time points (Days 3 and 4). Understanding the regulation of calbindin during cisplatin nephrotoxicity further enhances its utility as a potential urinary biomarker of kidney damage. The results of the current study support the combined use of a proximal (Kim-1) and distal tubule (calbindin) marker to phenotype acute kidney injury secondary to cisplatin administration.

During pregnancy, the migration and invasion of extravillous trophoblasts (EVTs) into the maternal uterus is essential for proper development of the placenta and fetus. During the first trimester, EVTs engraft and remodel maternal spiral arteries allowing for efficient blood flow and the transfer of essential nutrients and oxygen to the fetus. Aberrant migration of EVTs leading to either shallow or deep invasion into the uterus has been implicated in a number of gestational pathologies including preeclampsia, fetal growth restriction, and placenta accreta spectrum. The migration and invasion of EVTs is well-coordinated to ensure proper placentation. However, recent data point to the ability of xenobiotics to disrupt EVT migration. These xenobiotics include heavy metals, endocrine disrupting chemicals, and organic contaminants and have often been associated with adverse pregnancy outcomes. In most instances, xenobiotics appear to reduce EVT migration; however, there are select examples of enhanced motility after chemical exposure. In this review, we provide an overview of the 1) current experimental approaches used to evaluate EVT migration and invasion in vitro, 2) ability of environmental chemicals and pharmaceuticals to enhance or retard EVT motility, and 3) signaling pathways responsible for altered EVT migration that are sensitive to disruption by xenobiotics.

Traditional experimental approaches to evaluate hepatotoxicity are expensive and time-consuming. As an advanced framework of risk assessment, adverse outcome pathways (AOPs) describe the sequence of molecular and cellular events underlying chemical toxicities. We aimed to develop an AOP that can be used to predict hepatotoxicity by leveraging computational modeling and in vitro assays. We curated 869 compounds with known hepatotoxicity classifications as a modeling set and extracted assay data from PubChem. The antioxidant response element (ARE) assay, which quantifies transcriptional responses to oxidative stress, showed a high correlation to hepatotoxicity (PPV=0.82). Next, we developed quantitative structure-activity relationship (QSAR) models to predict ARE activation for compounds lacking testing results. Potential toxicity alerts were identified and used to construct a mechanistic hepatotoxicity model. For experimental validation, 16 compounds in the modeling set and 12 new compounds were selected and tested using an in-house ARE-luciferase assay in HepG2-C8 cells. The mechanistic model showed good hepatotoxicity predictivity (accuracy = 0.82) for these compounds. Potential false positive hepatotoxicity predictions by only using ARE results can be corrected by incorporating structural alerts and vice versa. This mechanistic model illustrates a potential toxicity pathway for hepatotoxicity, and this strategy can be expanded to develop predictive models for other complex toxicities.

Background: Vancomycin is associated with nephrotoxicity and the mechanism may in part be related to oxidative stress. In vitro and preclinical studies suggest melatonin supplementation decreases oxidative stress. The objective of this study was to evaluate concomitant use of melatonin and vancomycin and the incidence of acute kidney injury (AKI). Methods: We performed a retrospective cohort study at a large community medical center. All consecutive patients admitted to the medical center between January 2016 and September 2020 who received vancomycin therapy alone or concomitantly with melatonin as part of ordinary care were considered for inclusion. The primary endpoint was the development of AKI defined as an absolute increase in serum creatinine of ≥ 0.3 mg/dL or a ≥ 50% increase in serum creatinine. All data were analyzed using descriptive statistics. A multivariable logistic regression was constructed to account for potential confounding variables. Results: A total of 303 adult patients meeting inclusion and exclusion criteria treated with vancomycin were identified, 101 of which received melatonin concomitantly. Overall baseline characteristics were similar between the two groups except for the incidence of bactremia/sepsis. After controlling for vancomycin area under the curve, baseline creatinine clearance, and intensive care unit admission in multivariable logistic regression, melatonin use was associated with a 63% decrease in AKI (odds ratio [OR], 0.37; 95% confidence interval [CI], 0.14 - 0.96; p =0.041). Conclusions: Melatonin use was associated with a significant reduction in vancomycin-related AKI. Although this was a retrospective study with a small sample size, given the magnitude of the difference seen, further large prospective studies are warranted.

The organic cation transporter 2 (OCT2) and multidrug and toxin extrusion protein 1 (MATE1) mediate the renal secretion of drugs. Recent studies suggest that ondansetron, a 5-HT3 antagonist drug used to prevent nausea and vomiting, can inhibit OCT2- and MATE1-mediated transport. The purpose of this study was to test the ability of five 5-HT3 antagonist drugs to inhibit the OCT2 and MATE1 transporters. The transport of the OCT2/MATE1 probe substrate ASP+ was assessed using two models: (1) HEK293 kidney cells overexpressing human OCT2 or MATE1, and (2) MDCK cells transfected with human OCT2 and MATE1. In HEK293 cells, the inhibition of ASP+ uptake by OCT2 listed in order of potency was palonosetron (IC50: 2.6 μM) &gt, ondansetron &gt, granisetron &gt, tropisetron &gt, dolasetron (IC50: 85.4 μM) and the inhibition of ASP+ uptake by MATE1 in order of potency was ondansetron (IC50: 0.1 μM) &gt, palonosetron = tropisetron &gt, dolasetron (IC50: 27.4 μM). Ondansetron (0.5–20 μM) inhibited the basolateral-to-apical transcellular transport of ASP+ up to 64%. Higher concentrations (10 and 20 μM) of palonosetron, tropisetron, and dolasetron similarly reduced the transcellular transport of ASP+. In double-transfected OCT2-MATE1 MDCK cells, ondansetron at concentrations of 0.5 and 2.5 μM caused significant intracellular accumulation of ASP+. Taken together, these data suggest that 5-HT3 antagonist drugs may inhibit the renal secretion of cationic drugs by interfering with OCT2 and/or MATE1 function.

The protein–protein interaction (PPI) between kelch-like ECH-associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor 2 (Nrf2) is recognized as a promising target for the prevention and treatment of oxidative stress-related inflammatory diseases. Herein, a series of novel 1,4-bis(arylsulfonamido)naphthalene-N,N'-diacetic acid analogs (7p-t and 8c) were designed to further explore the structure–activity relationships of the series. Their activities were measured first with a fluorescence polarization (FP) assay and more potent compounds were further evaluated using a more sensitive time-resolved fluorescence energy transfer (TR-FRET) assay, demonstrating IC50 values between 7.2 and 31.3 nM. In cytotoxicity studies, the naphthalene derivatives did not show noticeable toxicity to human HepG2-C8 and mouse brain BV-2 microglia cells. Among them, compound 7q bearing oxygen-containing fused rings was shown to significantly stimulate the cellular Nrf2 signaling pathway, including activation of antioxidant response element (ARE)-controlled expression of Nrf2 target genes and proteins. More importantly, 7q suppressed up-regulation of several pro-inflammatory cytokines in lipopolysaccharide (LPS)-challenged BV-2 microglial cells, representing a potential therapeutic application for controlling neuroinflammatory disorders.

In the present work, in vivo transporter knockout (KO) mouse models were used to characterize the disposition of diclofenac (DCF) and its primary metabolites following a single sub-toxic dose in mice lacking breast cancer resistance protein (Bcrp) or multidrug resistance-associated protein (Mrp)3. The results indicate that Bcrp acts a canalicular efflux mediator for DCF as wild-type (WT) mice had biliary excretion values that were 2.2- to 2.6-fold greater than Bcrp KO mice, though DCF plasma levels were not affected. The loss of Bcrp resulted in a 1.8- to 3.2-fold increase of diclofenac acyl glucuronide (DCF-AG) plasma concentrations in KO animals compared to WT mice, while the biliary excretion of DCF-AG increased 1.4-fold in WT versus KO mice. Furthermore, Mrp3 was found to mediate the basolateral transport of DCF-AG, but not DCF or 4ʹ-hydroxy diclofenac. WT mice had DCF-AG plasma concentrations 7.0- to 8.6-fold higher than Mrp3 KO animals, however there were no changes in biliary excretion of DCF-AG. Vesicular transport experiments with human MRP3 demonstrated that MRP3 is able to transport DCF-AG via a low and high affinity binding site. The low affinity MRP3 transport had a Vmax and Km of 170 pmol/min/mg and 98.2 μM, respectively, while the high affinity Vmax and Km parameters were estimated to be 71.9 pmol/min/mg and 1.78 μ;M, respectively. In summary, we offer evidence that the disposition of DCF-AG can be affected by both Bcrp and Mrp3, and these findings may be applicable to humans. SIGNIFICANCE STATEMENT We report that diclofenac was found to be a substrate of Bcrp but not Mrp3 using mouse transporter models. Using the same models, diclofenac acyl glucuronide was found to be a substrate of both Bcrp and Mrp3. Importantly, the loss of Mrp3 affects diclofenac acyl glucuronide plasma concentrations but has no apparent effect with respect to liver concentrations or biliary excretion rates. We also report that diclofenac acyl glucuronide as an apparent low affinity and high affinity for human MRP3 compared to human MRP2 for which a single binding site is considered operative.

In the placenta, the breast cancer resistance protein (BCRP)/ABCG2 efflux transporter limits the maternal-to-fetal transfer of drugs and chemicals. Previous research has pointed to the estrogenic mycotoxin zearalenone as a potential substrate for BCRP. Here, we sought to assess the role of the BCRP transporter in the transplacental disposition of zearalenone during pregnancy. In vitro transwell transport assays employing BCRP/Bcrp-transfected Madine-Darby canine kidney cells and BeWo trophoblasts with reduced BCRP expression were used to characterize the impact of BCRP on the bidirectional transport of zearalenone. In both models, the presence of BCRP protein increased the basolateral-to-apical transport and reduced the apical-to-basolateral transport of zearalenone over a 2-h period. In vivo pharmacokinetic analyses were then performed using pregnant wild-type and Bcrp(−/−) mice after a single tail vein injection of zearalenone. Zearalenone and its metabolite α-zearalenol were detectable in serum, placentas, and fetuses from all animals, and β-zearalenol was detected in serum and fetuses, but not placentas. There were no significant differences in the maternal serum concentrations of any analytes between the two genotypes. In Bcrp(−/−) mice, the free fetal concentrations of zearalenone, α-zearalenol, and β-zearalenol were increased by 115%, 84%, and 150%, respectively, when compared with wild-type mice. Concentrations of free zearalenone and α-zearalenol were elevated 145% and 78% in Bcrp(−/−) placentas, respectively, when compared with wild-type placentas. Taken together, these data indicate that the placental BCRP transporter functions to reduce the fetal accumulation of zearalenone, which may impact susceptibility to developmental toxicities associated with in utero zearalenone exposure.

NADH cytochrome b(5) reductase mediates electron transfer from NADH to cytochrome b(5) utilizing flavin adenine dinucleotide as a redox cofactor. Reduced cytochrome b(5) is an important cofactor in many metabolic reactions including cytochrome P450-mediated xenobiotic metabolism, steroid biosynthesis and fatty acid metabolism, hemoglobin reduction, and methionine and plasmalogen synthesis. Using recombinant human enzyme, we discovered that cytochrome b5 reductase mediates redox cycling of a variety of quinones generating superoxide anion, hydrogen peroxide, and, in the presence of transition metals, hydroxyl radicals. Redox cycling activity was oxygen-dependent and preferentially utilized NADH as a co-substrate; NADH was 5–10 times more active than NADPH in supporting redox cycling. Redox cycling activity was greatest for 9,10-phenanthrenequinone and 1,2-naphthoquinone, followed by 1,4-naphthoquinone and 2-methyl-1,4-naphthoquinone (menadione), nitrofurantoin and 2-hydroxyestradiol. Using menadione as the substrate, quinone redox cycling was found to inhibit reduction of cytochrome b(5) by cytochrome b(5) reductase, as measured by heme spectral changes in cytochrome b(5). Under anaerobic conditions where redox cycling is inhibited, menadione had no effect on the reduction of cytochrome b(5). Chemical redox cycling by cytochrome b(5) reductase may be important in generating cytotoxic reactive oxygen species in target tissues. This activity, together with the inhibition of cytochrome b(5) reduction by redox-active chemicals and consequent deficiencies in available cellular cytochrome b(5), are likely to contribute to tissue injury following exposure to quinones and related redox active chemicals.

The placenta is essential for regulating the exchange of solutes between the maternal and fetal circulations. As a result, the placenta offers support and protection to the developing fetus by delivering crucial nutrients and removing waste and xenobiotics. ATP-binding cassette transporters, including multidrug resistance protein 1, multidrug resistance-associated proteins, and breast cancer resistance protein, remove chemicals through active efflux and are considered the primary transporters within the placental barrier. Altered transporter expression at the barrier could result in fetal exposure to chemicals and/or accumulation of xenobiotics within trophoblasts. Emerging data demonstrate that expression of these transporters is changed in women with pregnancy complications, suggesting potentially compromised integrity of placental barrier function. The purpose of this review is to summarize the regulation of placental efflux transporters during medical complications of pregnancy, including 1) placental inflammation/infection and chorioamnionitis, 2) hypertensive disorders of pregnancy, 3) metabolic disorders including gestational diabetes and obesity, and 4) fetal growth restriction/altered fetal size for gestational age. For each disorder, we review the basic pathophysiology and consider impacts on the expression and function of placental efflux transporters. Mechanisms of transporter dysregulation and implications for fetal drug and toxicant exposure are discussed. Understanding how transporters are up- or downregulated during pathology is important in assessing possible exposures of the fetus to potentially harmful chemicals in the environment as well as the disposition of novel therapeutics intended to treat placental and fetal diseases. SIGNIFICANCE STATEMENT: Diseases of pregnancy are associated with reduced expression of placental barrier transporters that may impact fetal pharmacotherapy and exposure to dietary and environmental toxicants.

Multidrug resistance protein 1 (MDR1, ABCB1, P-glycoprotein) and breast cancer resistance protein (BCRP, ABCG2) are key efflux transporters that mediate the extrusion of drugs and toxicants in cancer cells and healthy tissues, including the liver, kidneys, and the brain. Altering the expression and activity of MDR1 and BCRP influences the disposition, pharmacodynamics, and toxicity of chemicals, including a number of commonly prescribed medications. Histone acetylation is an epigenetic modification that can regulate gene expression by changing the accessibility of the genome to transcriptional regulators and transcriptional machinery. Recently, studies have suggested that pharmacological inhibition of histone deacetylases (HDACs) modulates the expression and function of MDR1 and BCRP transporters as a result of enhanced histone acetylation. This review addresses the ability of HDAC inhibitors to modulate the expression and the function of MDR1 and BCRP transporters and explores the molecular mechanisms by which HDAC inhibition regulates these transporters. While the majority of studies have focused on histone regulation of MDR1 and BCRP in drug-resistant and drug-sensitive cancer cells, emerging data point to similar responses in nonmalignant cells and tissues. Elucidating epigenetic mechanisms regulating MDR1 and BCRP is important to expand our understanding of the basic biology of these two key transporters and subsequent consequences on chemoresistance as well as tissue exposure and responses to drugs and toxicants. SIGNIFICANCE STATEMENT: Histone deacetylase inhibitors alter the expression of key efflux transporters multidrug resistance protein 1 and breast cancer resistance protein in healthy and malignant cells.

Advancing a new drug to market requires substantial investments in time as well as financial resources. Crucial bioactivities for drug candidates, including their efficacy, pharmacokinetics (PK), and adverse effects, need to be investigated during drug development. With advancements in chemical synthesis and biological screening technologies over the past decade, a large amount of biological data points for millions of small molecules have been generated and are stored in various databases. These accumulated data, combined with new machine learning (ML) approaches, such as deep learning, have shown great potential to provide insights into relevant chemical structures to predict in vitro, in vivo, and clinical outcomes, thereby advancing drug discovery and development in the big data era.

PURPOSE: The ability to predict and detect clinical and subclinical nephrotoxicity early in the course of therapy has the potential to improve long-term outcomes in cancer patients receiving cisplatin chemotherapy. Pharmacokinetic parameters could serve as predictors of cisplatin-induced nephrotoxicity. METHODS: Participants [n=13] were treated with a one-hour cisplatin infusion [30–75 mg/m(2)]. Blood was collected pre-dose and up to 6 h post-dose. Urinary biomarkers [KIM-1, calbindin, clusterin, GST-pi, β2M, albumin, NGAL, osteopontin, clusterin, MCP-1, cystatin C and TFF3] were measured at baseline, day 3 and day 10. Total and unbound platinum concentrations were measured using ICP/MS. Noncompartmental analysis was performed and correlation and regression analyses evaluated the relationships between platinum pharmacokinetics and nephrotoxicity. RESULTS: Peak platinum urinary concentrations correlated with urinary levels of KIM-1, calbindin, clusterin, GST-pi, β2M, albumin, NGAL, osteopontin, clusterin, cystatin C and TFF3 at day 10. Unbound platinum plasma concentrations at 2 h also correlated with urinary clusterin, β2M, cystatin C, NGAL, osteopontin, and TFF3 at day 3. Regression analyses suggested 2 h total plasma platinum concentrations greater than 2,000 ng/ml and peak urinary platinum concentrations above 24,000 ng/ml may serve as potential approximations for elevated risk of nephrotoxicity. Platinum area under the plasma concentration time curve was associated with serum creatinine and estimated glomerular filtration rate. CONCLUSIONS: Peak plasma and urinary platinum concentrations and pharmacokinetic parameters were associated with risk of subclinical cisplatin-induced kidney injury as assessed using novel urinary biomarkers. Future studies will examine these relationships in larger clinical populations of cisplatin-induced acute kidney injury.

In most mammals, labor is heralded by the withdrawal of progesterone. In humans, circulating progesterone levels increase as gestation advances while placental expression of progesterone receptor A (PR-A) declines. As a result of PR-A downregulation, the non-canonical NF-κB pathway is activated, an event implicated in triggering labor. Here, we sought to identify fetal-derived mediator(s) that represses placental PR-A in human placenta leading to activation of pro-labor signaling. Lipidomic profiling demonstrated enrichment of platelet-activating factor (PAF) in exosomes originating from the human fetus. Exposure of primary cytotrophoblasts to fetal exosomes from term pregnancies reduced PR-A expression by > 50%, and PAF also reduced PR-A message levels in a dose-dependent manner. Notably, fetal exosomes from preterm pregnancies had lower PAF levels and no effect on PR-A expression. Synthetic PAF–induced DNA methylation increases by 20% at the PR-A promoter, leading to recruitment of corepressors and downregulation of PR-A in cytotrophoblast. Furthermore, suppression of PR-A by PAF-stimulated expression of the pro-labor genes, corticotropin-releasing hormone (CRH) and cyclooxygenase-2 (COX-2), which was reversed by disruption of the DNA methyltransferases 3B and 3L. Taken together, PAF represents a novel fetal–derived candidate for initiation of labor by stimulating methylation and repression of PR-A and activating pro-labor signaling in trophoblast.

Graphical abstract, Highlights • KIM-1, calbindin, and TFF3 are increased in urine of patients prescribed cisplatin. • KIM-1 concentrations remain elevated with subsequent cisplatin treatment cycles. • Calbindin concentrations increased only during initial cycles of cisplatin chemotherapy. • TFF3 increased similarly on both cycles and returned to baseline in between., Proteins secreted into urine following tubular injury are being increasingly used as biomarkers of clinical and subclinical nephrotoxicity. In the present study, we sought to characterize the time-dependent urinary excretion of three promising biomarkers, kidney injury molecule-1 (KIM-1), calbindin, and trefoil factor 3 (TFF3), during two different chemotherapy cycles in 27 patients with solid tumors prescribed the anticancer drug cisplatin (≥25 mg/m2). Urinary biomarkers were evaluated at Days 3 and 10 during an initial and a subsequent cycle of cisplatin chemotherapy. Longitudinal analyses compared the mean difference estimations for biomarker concentrations during and across the initial and subsequent cycles of cisplatin treatment. Traditional biomarkers including serum creatinine, estimated glomerular filtration rate, and blood urea nitrogen were unchanged during and across both cycles of cisplatin therapy. In response to the initial cycle, urinary KIM-1 concentrations increased from baseline and remained elevated through a subsequent cycle of cisplatin chemotherapy. By comparison, urinary levels of calbindin were elevated 10 days after the initial cisplatin treatment, but largely unchanged by cisplatin exposure in a subsequent cycle. Early elevations in urinary TFF3 at 3 days after cisplatin administration were observed consistently in both the initial and subsequent cycle of cisplatin treatment. In conclusion, the longitudinal assessment of biomarker performance in the same cohort of oncology patients reveals different patterns of urinary excretion between initial and subsequent cycles of cisplatin-containing chemotherapy. These data add novel cycle-dependent insight to the growing literature addressing the ability of urinary biomarkers to detect subclinical renal injury in patients receiving cisplatin.

The breast cancer resistance protein (BCRP/ABCG2) is a maternally-facing efflux transporter that regulates the placental disposition of chemicals. Transcription factors and gene variants are important regulatory factors that influence transporter expression. In this study, we sought to identify the genetic and transcriptional mechanisms underlying the interindividual expression of BCRP mRNA and protein across 137 term placentas from uncomplicated pregnancies. Placental expression of BCRP and regulatory transcription factor mRNAs was measured using multiplex-branched DNA analysis. BCRP expression and ABCG2 genotypes were determined using Western blot and Fluidigm Biomark genetic analysis, respectively. Placentas were obtained from a racially and ethnically diverse population, including Caucasian (33%), African American (14%), Asian (14%), Hispanic (15%), and mixed (16%) backgrounds, as well as unknown origins (7%). Between placentas, BCRP mRNA and protein varied up to 47-fold and 14-fold, respectively. In particular, BCRP mRNA correlated significantly with known transcription factor mRNAs, including nuclear factor erythroid 2-related factor 2 and aryl hydrocarbon receptor. Somewhat surprisingly, single-nucleotide polymorphisms (SNPs) in the ABCG2 noncoding regions were not associated with variation in placental BCRP mRNA or protein. Instead, the coding region polymorphism (C421A/Q141K) corresponded with 40%–50% lower BCRP protein in 421C/A and 421A/A placentas compared with wild types (421C/C). Although BCRP protein and mRNA expression weakly correlated (r = 0.25, P = 0.040), this relationship was absent in individuals expressing the C421A variant allele. Study results contribute to our understanding of the interindividual regulation of BCRP expression in term placentas and may help to identify infants at risk for increased fetal exposure to chemicals due to low expression of this efflux protein.

The breast cancer resistance protein (BCRP/ABCG2) is an efflux transporter in the placental barrier. By transporting chemicals from the fetal to the maternal circulation, BCRP limits fetal exposure to a range of drugs, toxicants, and endobiotics such as bile acids and hormones. The purpose of the present studies was to 1) determine whether BCRP localizes to highly-ordered, cholesterol-rich lipid raft microdomains in placenta microvillous membranes, and 2) determine the impact of cholesterol on BCRP-mediated placental transport in vitro.BCRP expression was analyzed in lipid rafts isolated from placentas from healthy, term pregnancies and BeWo trophoblasts by density gradient ultracentrifugation. BeWo cells were also tested for their ability to efflux BCRP substrates after treatment with the cholesterol sequestrant methyl-β-cyclodextrin (MβCD, 5 mM, 1 h) or the cholesterol synthesis inhibitor pravastatin (200 μM, 48 h).BCRP was found to co-localize with lipid raft proteins in detergent-resistant, lipid raft-containing fractions from placental microvillous membranes and BeWo cells. Treatment of BeWo cells with MβCD redistributed BCRP protein into higher density non-lipid raft fractions. Repletion of the cells with cholesterol restored BCRP localization to lipid raft-containing fractions. Treatment of BeWo cells with MβCD or pravastatin increased cellular retention of two BCRP substrates, the fluorescent dye Hoechst 33342 and the mycotoxin zearalenone. Repletion with cholesterol restored BCRP transporter activity. Taken together, these data demonstrate that cholesterol may play a critical role in the post-translational regulation of BCRP in placental lipid rafts.

The success of cisplatin-containing regimens to treat solid tumors is limited, in part, by nephrotoxicity. In rodents, several urinary proteins have emerged that are sensitive indicators of cisplatin-induced kidney injury. We sought to characterize time-dependent changes in the urinary concentrations of 12 proteins, including kidney injury molecule-1 (KIM-1), calbindin, beta 2-microglobulin (β2M), and trefoil factor 3 (TFF3) after cisplatin therapy. Urine was collected at baseline, 3 days (range, 2-5 days), and 10 days (range, 9-11 days) from 57 patients with solid tumors receiving outpatient cisplatin therapy (≥25 mg/m2 ). Serum creatinine was largely unchanged after cisplatin infusion. However, compared with baseline values, several novel biomarkers were significantly increased in the urine, including β2M, which was threefold higher by day 3 (P < 0.0001). Urinary KIM-1 and TFF3 were elevated twofold by day 10 (P = 0.002 and P = 0.002, respectively), whereas calbindin levels were increased eightfold (P < 0.0001). We report novel time-dependent changes in the urinary excretion of noninvasive markers of subclinical kidney injury after cisplatin treatment.

The farnesoid X receptor (Fxr) controls bile acid homeostasis by coordinately regulating the expression of synthesizing enzymes (Cyp7a1, Cyp8b1), conjugating enzymes (Bal, Baat) and transporters in the ileum (Asbt, Ostα/β) and liver (Ntcp, Bsep, Ostβ). Transcriptional regulation by Fxr can be direct, or through the ileal Fgf15/FGF19 and hepatic Shp pathways. Circulating bile acids are increased during pregnancy due to hormone-mediated disruption of Fxr signaling. While this adaptation enhances lipid absorption, elevated bile acids may predispose women to develop maternal cholestasis. The objective of this study was to determine whether short-term treatment of pregnant mice with GW4064 (a potent FXR agonist) restores Fxr signaling to the level observed in virgin mice. Plasma, liver and ilea were collected from virgin and pregnant mice administered vehicle or GW4064 by oral gavage. Treatment of pregnant mice with GW4064 induced ileal Fgf15, Shp and Ostα/β mRNAs, and restored hepatic Shp, Bal, Ntcp, and Bsep back to vehicle-treated virgin levels. Pregnant mice exhibited 2.5-fold increase in Cyp7a1 mRNA compared to virgin controls, which was reduced by GW4064. Similarly treatment of mouse primary hepatocytes with plasma isolated from pregnant mice induced Cyp7a1 mRNA by nearly 3-fold as compared to virgin plasma, which could be attenuated by co-treatment with either GW4064 or recombinant FGF19 protein. Collectively, these data reveal that repressed activity of intestinal and hepatic Fxr in pregnancy, as previously demonstrated, may be restored by pharmacological activation. This study provides the basis for a novel approach to restore bile acid homeostasis in patients with maternal cholestasis.