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.

Integrating Concentration-Dependent Toxicity Data and Toxicokinetics To Inform Hepatotoxicity Response Pathways.

Failure of animal models to predict hepatotoxicity in humans has created a push to develop biological pathway-based alternatives, such as those that use in vitro assays. Public screening programs (e.g., ToxCast/Tox21 programs) have tested thousands of chemicals using in vitro high-throughput screening (HTS) assays. Developing pathway-based models for simple biological pathways, such as endocrine disruption, has proven successful, but development remains a challenge for complex toxicities like hepatotoxicity, due to the many biological events involved. To this goal, we aimed to develop a computational strategy for developing pathway-based models for complex toxicities. Using a database of 2171 chemicals with human hepatotoxicity classifications, we identified 157 out of 1600+ ToxCast/Tox21 HTS assays to be associated with human hepatotoxicity. Then, a computational framework was used to group these assays by biological target or mechanisms into 52 key event (KE) models of hepatotoxicity. KE model output is a KE score summarizing chemical potency against a hepatotoxicity-relevant biological target or mechanism. Grouping hepatotoxic chemicals based on the chemical structure revealed chemical classes with high KE scores plausibly informing their hepatotoxicity mechanisms. Using KE scores and supervised learning to predict in vivo hepatotoxicity, including toxicokinetic information, improved the predictive performance. This new approach can be a universal computational toxicology strategy for various chemical toxicity evaluations.

Data-Driven Quantitative Structure-Activity Relationship Modeling for Human Carcinogenicity by Chronic Oral Exposure.

Traditional methodologies for assessing chemical toxicity are expensive and time-consuming. Computational modeling approaches have emerged as low-cost alternatives, especially those used to develop quantitative structure-activity relationship (QSAR) models. However, conventional QSAR models have limited training data, leading to low predictivity for new compounds. We developed a data-driven modeling approach for constructing carcinogenicity-related models and used these models to identify potential new human carcinogens. To this goal, we used a probe carcinogen dataset from the US Environmental Protection Agency's Integrated Risk Information System (IRIS) to identify relevant PubChem bioassays. Responses of 25 PubChem assays were significantly relevant to carcinogenicity. Eight assays inferred carcinogenicity predictivity and were selected for QSAR model training. Using 5 machine learning algorithms and 3 types of chemical fingerprints, 15 QSAR models were developed for each PubChem assay dataset. These models showed acceptable predictivity during 5-fold cross-validation (average CCR = 0.71). Using our QSAR models, we can correctly predict and rank 342 IRIS compounds' carcinogenic potentials (PPV = 0.72). The models predicted potential new carcinogens, which were validated by a literature search. This study portends an automated technique that can be applied to prioritize potential toxicants using validated QSAR models based on extensive training sets from public data resources.

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.

Regulation of Placental Efflux Transporters during Pregnancy Complications.

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.

Regulation of renal calbindin expression during cisplatin-induced kidney injury.

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.

Predictive modeling of estrogen receptor agonism, antagonism, and binding activities using machine- and deep-learning approaches.

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 ERß) 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.

Renoprotective Effects of Melatonin against Vancomycin-Related Acute Kidney Injury in Hospitalized Patients: a Retrospective Cohort Study.

Vancomycin is associated with nephrotoxicity, and the mechanism may in part be related to oxidative stress. In vitro and preclinical studies suggest that 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). 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. We identified a total of 303 adult patients meeting inclusion and exclusion criteria treated with vancomycin, 101 of which received melatonin concomitantly. Overall baseline characteristics were similar between the two groups except for the incidence of bacteremia/sepsis. After controlling for the vancomycin area under the curve, baseline creatinine clearance, and intensive care unit admission in a multivariable logistic regression analysis, melatonin use was associated with a 63% decrease in AKI (odds ratio [OR], 0.37; 95% confidence interval [CI], 0.14 to 0.96; P = 0.041). 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.

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.

Revealing Adverse Outcome Pathways from Public High-Throughput Screening Data to Evaluate New Toxicants by a Knowledge-Based Deep Neural Network Approach.

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 ß (ERα and ERß) binding potentials. The target activity was rodent uterotrophic bioactivity driven by ERα/ERß activations. After training, the resultant network successfully inferred critical relationships among ERα/ERß 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/ERß 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.

Optimization of 1,4-bis(arylsulfonamido)naphthalene-N,N'-diacetic acids as inhibitors of Keap1-Nrf2 protein-protein interaction to suppress neuroinflammation.

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 Vitro Inhibition of Renal OCT2 and MATE1 Secretion by Antiemetic Drugs.

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) > ondansetron > granisetron > tropisetron > dolasetron (IC50: 85.4 µM) and the inhibition of ASP+ uptake by MATE1 in order of potency was ondansetron (IC50: 0.1 µM) > palonosetron = tropisetron > granisetron > 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.

Epigenetic Regulation of Multidrug Resistance Protein 1 and Breast Cancer Resistance Protein Transporters by Histone Deacetylase Inhibition.

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.

Identification and Characterization of Efflux Transporters That Modulate the Subtoxic Disposition of Diclofenac and Its Metabolites.

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 subtoxic dose in mice lacking breast cancer resistance protein (Bcrp) or multidrug resistance-associated protein (Mrp)3. The results indicate that Bcrp acts as 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, although 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 with 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 4'-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 low- and high-affinity binding sites. The low-affinity MRP3 transport had a V max and K m of 170 pmol/min/mg and 98.2 µM, respectively, while the high-affinity V max and K m parameters were estimated to be 71.9 pmol/min/mg and 1.78 µM, 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.

Anandamide down-regulates placental transporter expression through CB2 receptor-mediated inhibition of cAMP synthesis.

The BCRP/ABCG2 efflux transporter is expressed on the membrane of placental syncytiotrophoblasts and protects the fetus from toxicant exposure. Syncytiotrophoblasts arise from the fusion of cytotrophoblasts, a process negatively regulated by the endocannabinoid, anandamide (AEA). It is unknown whether AEA can influence fetal concentrations of xenobiotics by modulating the expression of transporters in syncytiotrophoblasts. Here, we sought to characterize and identify the mechanism(s) responsible for AEA-mediated down-regulation of the BCRP transporter in human placental explants and BeWo trophoblasts. Treatment of human placental explants with AEA (1 µM, 24 h) reduced hCGα, syncytin-1, and BCRP mRNAs by ˜30%. Similarly, treatment of BeWo trophoblasts with AEA (0-10 µM, 3-24 h) coordinately down-regulated mRNAs for hCGß, syncytin-2, and BCRP. In turn, AEA increased the sensitivity of trophoblasts to the cytotoxicity of mitoxantrone, a known BCRP substrate, and environmental and dietary contaminants including mycoestrogens and perfluorinated chemicals. AEA-treated trophoblasts also demonstrated reduced BCRP transport of the mycoestrogen zearalenone and the diabetes drug glyburide, labeled with BODIPY. The AEA-mediated reduction of BCRP mRNA was abrogated when placental cells were co-treated with AM630, a CB2 receptor inhibitor, or 8-Br-cAMP, a cAMP analog. AEA reduced intracellular cAMP levels in trophoblasts by 75% at 1 h, and completely inhibited forskolin-induced phosphorylation of the cAMP response element binding protein (CREB). AEA also decreased p-CREB binding to the BCRP promoter. Taken together, our data indicate that AEA down-regulates placental transporter expression and activity via CB2-cAMP signaling. This novel mechanism may explain the repression of placental BCRP expression observed during diseases of pregnancy.

Brain region-specific regulation of histone acetylation and efflux transporters in mice.

Multidrug resistance protein 1 (MDR1) and breast cancer resistance protein (BCRP) protect the brain by restricting the passage of chemicals across the blood-brain barrier. Prior studies have demonstrated the epigenetic regulation of MDR1 and BCRP in cancer cells treated with histone deacetylase (HDAC) inhibitors that enhance histone acetylation and gene transcription. In the present study, we tested the in vivo effects of two HDAC inhibitors, valproic acid (VPA; 400 mg/kg) and apicidin (5 mg/kg), on Mdr1 and Bcrp transporter expression in brain regions of adult male mice injected intraperitoneally daily for 7 days. VPA increased Mdr1 protein expression in the striatum (70%) and Bcrp protein in the midbrain (30%). Apicidin enhanced striatal Mdr1 protein (30%) and hippocampal Bcrp protein (20%). Transporter induction correlated with increased histone H3 acetylation in discrete brain regions. In conclusion, HDAC inhibitors upregulate transporter proteins in vivo, which may be important in regulating regional xenobiotic disposition within the brain.

Interindividual Regulation of the Breast Cancer Resistance Protein/ABCG2 Transporter in Term Human Placentas.

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.

Quinone and nitrofurantoin redox cycling by recombinant cytochrome b5 reductase.

NADH cytochrome b5 reductase mediates electron transfer from NADH to cytochrome b5 utilizing flavin adenine dinucleotide as a redox cofactor. Reduced cytochrome b5 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 b5 by cytochrome b5 reductase, as measured by heme spectral changes in cytochrome b5. Under anaerobic conditions where redox cycling is inhibited, menadione had no effect on the reduction of cytochrome b5. Chemical redox cycling by cytochrome b5 reductase may be important in generating cytotoxic reactive oxygen species in target tissues. This activity, together with the inhibition of cytochrome b5 reduction by redox-active chemicals and consequent deficiencies in available cellular cytochrome b5, are likely to contribute to tissue injury following exposure to quinones and related redox active chemicals.

In Vitro Transport Activity and Trafficking of MRP2/ABCC2 Polymorphic Variants.

PURPOSE: Multidrug resistance-associated protein 2 (MRP2/ABCC2) is an efflux pump that removes drugs and chemicals from cells. We sought to characterize the expression, trafficking and in vitro activity of seven single nucleotide polymorphisms (SNPs) in the ABCC2 gene. METHODS: ABCC2 SNPs were generated using site-directed mutagenesis and stably expressed in Flp-In HEK293 cells, which allows targeted insertion of transgenes within the genome. Total and cell surface expression of MRP2 as well as accumulation of substrates (calcein AM and 5(6)-carboxy-2',7'-dichlorofluorescein diacetate, CDCF) were quantified in cells or inverted membrane vesicles expressing wild-type (WT) or variant forms. RESULTS: The cell surface expression of the C-24T-, G1249A-, G3542T-, T3563A-, C3972T- and G4544A-MRP2 variants was similar to WT-MRP2. While expression was similar, transport of calcein AM was enhanced in cells expressing the G3542T-, T3563A-, C3972T-, and G4544A-MRP2 variants. By comparison, cells expressing the C2366T-MRP2 variant had 40-50% lower surface expression, which increased the accumulation of calcein AM up to 3-fold. Accumulation of CDCF in inverted membrane vesicles expressing the C2366T-MRP2 variant was also reduced by 50%. In addition, the G1249A-MRP2 variant had decreased transport of CDCF. CONCLUSIONS: Taken together, these data demonstrate that genetic variability in the ABCC2 gene influences the in vitro expression, trafficking, and transport activity of MRP2.

Regulation of the placental BCRP transporter by PPAR gamma.

Identifying regulators of placental breast cancer resistance protein (BCRP) expression is critical as downregulation of this transporter may increase exposure of the fetus to xenobiotics. Here, we sought to test whether the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) regulates BCRP expression in the placenta. To test this, human BeWo placental choriocarcinoma cells were cultured with the PPARγ agonist rosiglitazone or the PPARγ antagonist T0070907 for 24 h. Messenger RNA (mRNA) expression of syncytialization markers, GCM1 and hCGß, as well as BCRP increased with PPARγ agonist treatment. Conversely, BCRP mRNA and protein expression decreased 30%-50% with PPARγ antagonist treatment. Rosiglitazone enhanced BCRP protein expression and transport activity, resulting in a 20% greater efflux of the substrate Hoechst 33342 compared with control cells. These results suggest that PPARγ can upregulate BCRP expression in the placenta, which may be important in understanding mechanisms that protect the fetus from xenobiotic exposure during development.