Supratim Choudhuri

Structure, Function, Expression, Genomic Organization, and Single Nucleotide Polymorphisms of Human ABCB1 (MDR1), ABCC (MRP), and ABCG2 (BCRP) Efflux Transporters:

The ATP-binding cassette (ABC) transporters constitute a large family of membrane proteins, which transport a variety of compounds through the membrane against a concentration gradient at the cost of ATP hydrolysis. Substrates of the ABC transporters include lipids, bile acids, xenobiotics, and peptides for antigen presentation. As they transport exogenous and endogenous compounds, they reduce the body load of potentially harmful substances. One by-product of such protective function is that they also eliminate various useful drugs from the body, causing drug resistance. This review is a brief summary of the structure, function, and expression of the important drug resistance–conferring members belonging to three subfamilies of the human ABC family; these are ABCB1 (MDR1/P-glycoprotein of subfamily ABCB), subfamily ABCC (MRPs), and ABCG2 (BCRP of subfamily ABCG), which are expressed in various organs. In the text, the transporter symbol that carries the subfamily name (such as ABCB1, ABCC1, etc.) is used interchangeably with the corresponding original names, such as MDR1/P-glycoprotein, MRP1, etc., respectively. Both nomenclatures are maintained in the text because both are still used in the transporter literature. This helps readers relate various names that they encounter in the literature. It now appears that P-glycoprotein, MRP1, MRP2, and BCRP can explain the phenomenon of multidrug resistance in all cell lines analyzed thus far. Also discussed are the gene structure, regulation of expression, and various polymorphisms in these genes. Because genetic polymorphism is thought to underlie interindividual differences, including their response to drugs and other xenobiotics, the importance of polymorphism in these genes is also discussed.

Characterization of Organic Anion Transporting Polypeptide 1b2-null Mice: Essential Role in Hepatic Uptake/Toxicity of Phalloidin and Microcystin-LR

The liver-specific importer organic anion transporting polypeptide 1b2 (Oatp1b2, Slco1b2, also known as Oatp4 and Lst-1) and its human orthologs OATP1B1/1B3 transport a large variety of chemicals. Oatp1b2-null mice were engineered by homologous recombination and their phenotype was characterized. Oatp1b2 protein was absent in livers of Oatp1b2-null mice. Oatp1b2-null mice develop normally and breed well. However, adult Oatp1b2-null mice had moderate conjugated hyperbilirubinemia. Compared with wild-types, Oatp1b2-null mice had similar hepatic messenger RNA expression of most transporters examined except a higher Oatp1a4 but lower organic anion transporter 2. Intra-arterial injection of the mushroom toxin phalloidin (an Oatp1b2-specific substrate identified in vitro) caused cholestasis in wild-type mice but not in Oatp1b2-null mice. Hepatic uptake of fluorescence-labeled phalloidin was absent in Oatp1b2-null mice. Three hours after administration of microcystin-LR (a blue-green algae toxin), the binding of microcystin-LR to hepatic protein phosphatase 1/2a was much lower in Oatp1b2-null mice compared with wild-type mice. In contrast, Oatp1b2-null mice were transiently protected from decrease in bile flow induced by estradiol-17beta-D-glucuronide, a common substrate for Oatps. Oatp1b2-null mice were completely resistant to the hepatotoxicity induced by phalloidin and microcystin-LR, but were similarly sensitive to alpha-amanitin-induced hepatotoxicity compared with wild-type mice. In conclusion, Oatp1b2-null mice display altered basic physiology and markedly decreased hepatic uptake/toxicity of phalloidin and microcystin-LR. Oatp1b2-null mice are useful in elucidating the role of Oatp1b2 and its human orthologs OATP1B1/1B3 in hepatic uptake and systemic disposition of toxic chemicals and therapeutic drugs.

Epigenetic targets of some toxicologically relevant metals: a review of the literature

The term epigenetics was coined in the context of developmental studies, but the meaning of the term has evolved over time. Epigenetic modulators of gene expression are now known to include DNA methylation, chromatin modifications and noncoding RNAs. The observation that epigenetic changes can be transmitted transgenerationally makes the science of epigenetics very relevant to the field of environmental and molecular toxicology. Heavy metals constitute an important class of environmental contaminants that have been known to influence gene expression directly by binding various metal response elements in the target gene promoters. Recent research suggests that metals can also influence gene expression through epigenetic mechanisms; this adds a new twist to the complexity of metal‐mediated gene expression. Here, we review recent studies that investigate the epigenetic, gene expression, and biological effects of various inorganic and organic forms of heavy metals, such as cadmium, arsenic, nickel, chromium, methylmercury, lead, copper and organotin compounds. Copyright

MOLECULAR TARGETS OF EPIGENETIC REGULATION AND EFFECTORS OF ENVIRONMENTAL INFLUENCES

The true understanding of what we currently define as epigenetics evolved over time as our knowledge on DNA methylation and chromatin modifications and their effects on gene expression increased. The current explosion of research on epigenetics and the increasing documentation of the effects of various environmental factors on DNA methylation, chromatin modification, as well as on the expression of small non-coding RNAs (ncRNAs) have expanded the scope of research on the etiology of various diseases including cancer. The current review briefly discusses the molecular mechanisms of epigenetic regulation and expands the discussion with examples on the role of environment, such as the immediate environment during development, in inducing epigenetic changes and modulating gene expression.

Organic anion–transporting polypeptide 1b2 (Oatp1b2) is important for the hepatic uptake of unconjugated bile acids: Studies in Oatp1b2-null mice†‡

The organic anion–transporting polypeptide 1b family (Oatp1b2 in rodents and OATP1B1/1B3 in humans) is liver‐specific and transports various chemicals into the liver. However, the role of the Oatp1b family in the hepatic uptake of bile acids (BAs) into the liver is unknown. Therefore, in Oatp1b2‐null mice, the concentrations of BAs in plasma, liver, and bile were compared with wild‐type (WT) mice. It was first determined that livers of the Oatp1b2‐null mice were not compensated by altered expression of other hepatic transporters. However, the messenger RNA of Cyp7a1 was 70% lower in the Oatp1b2‐null mice. Increased expression of fibroblast growth factor 15 in intestines of Oatp1b2‐null mice might be responsible for decreased hepatic expression of Cyp7a1 in Oatp1b2‐null mice. The hepatic concentration and biliary excretion of conjugated and unconjugated BAs were essentially the same in Oatp1b2‐null and WT mice. The serum concentration of taurine‐conjugated BAs was essentially the same in the two genotypes. In contrast, the serum concentrations of unconjugated BAs were 3‐45 times higher in Oatp1b2‐null than WT mice. After intravenous administration of cholate to Oatp1b2‐null mice, its clearance was 50% lower than in WT mice, but the clearance of taurocholate was similar in the two genotypes. Conclusion: This study indicates that Oatp1b2 has a major role in the hepatic uptake of unconjugated BAs. (HEPATOLOGY 2011.)

Induction of Hepatic Glutathione S-Transferases in Male Mice by Prototypes of Various Classes of Microsomal Enzyme Inducers

The underlying need for glutathione S-transferase (Gst) induction is thought to be an adaptive response to chemical stress within the cell. Classical microsomal enzyme inducers (MEIs) increase the expression of biotransformation enzymes (phase I and II) and transporters through transcription factors, such as the aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor (PPAR) alpha, and nuclear factor erythroid-derived 2-related factor 2 (Nrf2). The effects of MEIs on the induction of hepatic Gsts in mice have not been comprehensively characterized. The purpose of this study was to determine the effects of 15 MEIs on the mRNA expression of 19 mouse Gsts. Male C57BL/6 mice were treated with three different activators each for AhR, CAR, PXR, PPARalpha, and Nrf2. In general, the Gsts are readily induced. All five transcription factors appear to play a role in Gst induction. The Nrf2 activators induced most Gsts (10), followed by the CAR, PXR, and PPARalpha activators (6-7), whereas the AhR ligands induced the least (1). Clofibrate, a PPARalpha agonist, induced most of the Gsts; however, all three PPARalpha agonists decreased Gstp1/2 mRNA. None of the 15 inducers was able to increase or only minimally increased eight of the Gsts (Gsta3, Gstk1, Gstm6, Gsto1, Gstp1/2, Gstt3, Gstz1, and MGst1). Thus, the protection afforded by a ligand for one of these transcription factors will depend on the activator, as well as which Gst that detoxifies the chemicals of interest.

Small noncoding RNAs: biogenesis, function, and emerging significance in toxicology.

In recent years, the discovery of small ncRNAs (noncoding RNAs) has unveiled a slew of powerful riboregulators of gene expression. So far, many different types of small ncRNAs have been described. Of these, miRNAs (microRNAs), siRNAs (small interfering RNAs), and piRNAs (Piwi‐interacting RNAs) have been studied in more detail. A significant fraction of genes in most organisms and tissues is targets of these small ncRNAs. Because these tiny RNAs are turning out to be important regulators of gene and genome expression, their aberrant expression profiles are expected to be associated with cellular dysfunction and disease. In fact, an ever‐increasing number of studies have implicated miRNAs and siRNAs in human health and disease ranging from metabolic disorders to diseases of various organ systems as well as various forms of cancer. Nevertheless, despite the flurry of research on these small ncRNAs, many aspects of their biology still remain to be understood. The following discussion focuses on some aspects of the biogenesis and function of small ncRNAs with major emphasis on miRNAs since these are the most widespread endogenous small ncRNAs that have been called “micromanagers” of gene expression. Their emerging significance in toxicology is also discussed.

Cadmium accumulation and metallothionein expression in brain of mice at different stages of development

Cadmium accumulation and metallothionein (MT) expression were studied in brains of adult mice as well as at different stages of development. MT expression was also studied in the eye of adult mice as well as at different stages of development. Using northern blot analysis with total RNA, MT mRNAs were not detected in day 16 through day 18 embryos or in postnatal animals up to day 14. Detectable expression of MT-I, -II, and -III mRNAs was obtained in brains of 30- and 60-day-old-mice. The expression of MT-III mRNA appeared to be much stronger in adults (12 weeks old or more) than in 30- and 60-day-old animals. In contrast, there was similar expression of MT-I and -II in 30- and 60-day-old mice. Cd distribution to brain was found to decrease with age; the brains of 7-day-old mice contained about 4-times more Cd than that of adult mice. Thus, an inverse correlation was observed between MT expression and Cd accumulation in brain.

Species variation in hepatic metallothionein.

Metallothionein (MT) is a low-molecular-weight protein involved in the homeostasis of endogenous metals and in the detoxication of heavy metals. In humans, the levels of hepatic MT have been shown to be up to 100 times the levels found in rat and mouse liver. In order to further investigate this species difference in hepatic MT levels, hepatic MT was quantified in 15 species (human, monkey, dog, cat, cow, pig, sheep, goat, rabbit, chicken, hamster, rat, mice, guinea pig, and frog). Fresh liver was obtained from each species and MT was quantified by 2 different metal-saturation assays. Results from the Cd-heme and Ag-heme assays showed that human, dog, cat, pig, and goat had the highest hepatic MT levels (400-700 micrograms/g liver). Monkey, cow, and sheep had moderate hepatic MT levels (about 200 micrograms/g liver), while rodents (mouse, rat, hamster, guinea pig, and rabbit) had low hepatic MT levels (2-10 micrograms/g liver). Hepatic MT levels in non-mammals (chick and frog) were slightly higher than rodents (about 20 micrograms/g liver). Sephadex G-75 column elution volumes ranged from 1.7 to 1.8, which implies that MT from all species had approximately the same molecular weight and similar structure. Copper and zinc concentration in the cytosols were measured by atomic absorption spectrophotometry. Dog and cat had the highest levels of Cu (86 and 50 micrograms/g liver, respectively), and pig and hamster were lowest (about 10 micrograms/g liver). Human, dog, cat, and goat had the highest levels of zinc (approximately 40-50 micrograms/g liver) while hamster and guinea pig were lowest (approximately 15 micrograms/g liver). The results show that there is a marked species difference in hepatic MT concentrations with dog, cat, and human having the highest levels.

From Waddington’s epigenetic landscape to small noncoding RNA: some important milestones in the history of epigenetics research

The term epigenetics was coined in 1942 by C.H. Waddington in the context of studies on development. Since then, the meaning of epigenetics changed over time. In the beginning, epigenetics was viewed as a phenomenon above and beyond genetics. Epigenetic explanations were invoked when genetics could not explain a phenomenon. From the mid-seventies, the state of understanding started changing. Epigenetics has now morphed from a phenomenon to a branch of science whose molecular underpinnings are well understood. The current state of knowledge of epigenetics has evolved as our understanding of DNA methylation, chromatin modifications, and noncoding RNA, and their effects on gene expression increased. At this time in the annals of epigentics research, it is appropriate to revisit some of the important discoveries that have helped advance the field to its current state. This is a very brief review of some early discoveries, and by no means is a complete account of the history of epigenetics. In this review, the early history has also been emphasized in order to underscore the transformation of the science of epigenetics from a phenomenon to a modern field of intense research.