Persyaratan Umum & Khusus Penerimaan CPNS 2014

Persyaratan Umum & Khusus Penerimaan CPNS 2014 Posted: Juni 2, 2014 in Detik News, Info Jobs & Beasiswa 81 Persyaratan Umum dan Khusus CPNS 2014 Informasi Umum Pendaftaran CPNS 2014 dibuka untuk mengisi kekosongan lowongan di berbagai instansi pusat dan daerah sebanyak 100.000 formasi dengan alokasi sebagai berikut: Formasi yang akan diberikan untuk pelamar umum adalah sebanyak 60 ribu alokasi formasi dan total alokasi formasi ini diperuntukan untuk CPNS daerah dan CPNS pusat Formasi untuk PPPK adalah sebanyak 40.000 formasi, dengan alokasi untuk pemerintah daerah dan pemerintah pusat Lowongan CPNS yang disediakan adalah untuk formasi tenaga kesehatan, tenaga guru, tenaga teknis/administrasi dan lowongan khusus yang diberikan untuk putra putri Papua dan kalangan penyandang cacat (kalangan disable) Pendaftaran CPNS diselenggarakan secara online dengan tata cara pendaftaran yang bisa anda lihat di http://www.asncpns.com/2014/05/panduan-registrasi-daftar-online-cpns.html Pendaftaran CPNS akan dimulai pada akhir Juni 2014 di website pendaftaran CPNS yang telah ditentukan dan sebelumnya akan diinformasikan melalui Job Fair CPNS yang akan diselenggarakan selama 2 hari berturut turut di Hotel Sahid Jaya pada tanggal 16 Juni sampai dengan tanggal 17 Juni 2014. Setiap pelamar yang mengirimkan lamaran dalam satu Kementerian hanya diperkenankan untuk mengirimkan satu lamaran saja, dimana lamaran tersebut ditujukan hanya untuk satu unit kerja dan satu unit formasi saja. Bobot Soal CPNS antara PPPK dan Pelamar Umum adalah berbeda, mekanisme tes ujian CPNS keduanya bisa dilihat di link berikut http://www.asncpns.com/2014/05/perbedaan-soal-cpns-pelamar-umum-dan.html Proses Seleksi dilaksanakan dalam beberapa tahap, diantaranya adalah: Screening Administrasi Tes Kompetensi Dasar yang meliputi TWK, TIU dan kepribadian. Untuk lebih jelas silakan akses Referensi pembelajaran resmi CPNS berdasarkan Juklak Juknis CPNS Tes Kompetensi Bidang. Tes TKB CPNS diberikan setelah peserta lulus point no 2 sesuai passing grade yang telah ditentukan. Masing masing ujian Tes Kompetensi bidang CPNS bisa dilihat di link berikut http://www.paketlkit.com/2014/05/paket-lkit-tkb.html Wawancara Kompetensi. Wawancara ini diberikan pada saat peserta tes telah lulus tahap 1 sampai dengan tahap 3. Wawancara diberikan untuk mengukur kompetensi masing masing peserta dengan mengukur dan berpatokan pada Interviewing Technical Analysis Untuk mengikuti seluruh seleksi CPNS, para peserta tes TIDAK DIPUNGUT BIAYA apapun. Persyaratan Umum CPNS 2014 Persyaratan umum CPNS ini adalah merupakan persyaratan yang harus dipersiapkan dan dipenuhi oleh masing masing pelamar sebelum bisa mengikuti seleksi tes cpns. Warga Negara Indonesia (WNI) Berusia antara 18 tahun dan 35 tahun pada tanggal 1 Desember 2014 Bagi pelamar yang berusia lebih dari 35 tahun hanya dikhususkan untuk kebutuhan formasi PPPK dan atau berdasarkan kebutuhan khusus, dimana setiap pelamar telah memiliki masa kerja yang telah ditentukan. Sehat Jasmani dan Rohani Bebas NARKOBA Khusus untuk pelamar kalangan sarjana dan diploma, berijazah Sarjana (S1), Diploma Empat (D4), atau Diploma Tiga (D3) dari Perguruan Tinggi Negeri (PTN)/Perguruan Tinggi Swasta (PTS) dengan akreditasi bidang studi minimal B oleh Badan Akreditasi Nasional Perguruan Tinggi (BAN-PT) dan Perguruan Tinggi Luar Negeri yang telah mendapat Penyetaraan Ijazah luar Negeri dari Kementerian Pendidikan dan Kebudayaan. Pada saat mendaftar, pelamar telah memiliki ijazah Perguruan Tinggi untuk kalangan S1 dan D3, dan memiliki Ijazah SMA untuk pelamar dari tingkat SMA Sederajat Indeks Prestasi Kumulatif (IPK) minimal 2,75 bagi lulusan dari program studi dengan akreditasi A dan minimal 3,00 bagi lulusan dari program studi dengan akreditasi B. Berkelakuan Baik dan tidak pernah dihukum penjara atau kurungan berdasarkan putusan pengadilan yang memiliki kekuatan hukum tetap Tidak pernah diberhentikan dengan hormat tidak atas permintaan sendiri atau tidak dengan hormat sebagai PNS/Anggota TNI/POLRI atau diberhentikan tidak dengan hormat sebagai pegawai swasta. Tidak sedang menjalani perjanjian/ikatan dinas/kontrak kerja pada instansi lain Persyaratan Khusus CPNS 2014Persyaratan khusus adalah persyaratan tambahan lainnya yang diberikan oleh masing masing instansi pemerintahan baik pusat ataupun daerah. Persayaratan khusus antara instansi satu dengan instansi lainnya akan berbeda. Beberapa Persyaratan khusus yangt biasa dipersyaratkan kepada pelamar diantaranya adalah: TOEFL/TOEFL Prediction dari Lembaga Bahasa Perguruan Tinggi Negeri atau UPT Bahasa Perguruan Tinggi Negeri atau lembaga pendidikan Bahasa Inggris terakreditasi dengan nilai minimal 450. TOEFL®IBT (Internet-Based TOEFL) yang diterbitkan oleh The Indonesian International Education Foundation (IIEF) dengan nilai minimal 53. IELTS yang diterbitkan oleh Yayasan Pendidikan Australia (IDP) dengan nilai minimal 5. Tata Cara Pendaftaran Pelamar wajib memiliki email aktif untuk bisa mengikuti proses rekrutment CPNS 2014 Pendaftaran dilakukan secara online, dengan tata cara yang telah disebutkan diatas Sebelum melakukan pendaftaran, silakan anda sediakan hal hal berikut ini Fotokopi KTP Legalisir Ijazah Legalisir Transkript Nilai Scan pas foto terbaru anda Setelah menyelesaikan pendaftaran secara online, peserta wajib mengirimkan semua berkas persyaratan yang ditentukan ditambah dengan print out asli bukti pendaftaran secara online yang telah ditandatangani oleh pelamar. Berkas yang akan dikirimkan disertai juga dengan SKCK dari Pihak Kepolisian Surat Keterangan Sehat dari Dokter Pemerintah Dokumen lain yang ditentukan oleh masing masing instansi Ketentuan Lain Tes CPNS 2014 menggunakan sistem CAT CPNS Tempat pelaksanaan tes dilaksanakan di tempat yang telah ditentukan. Informasi tentang lokasi tes dapat dilihat di laman berikut http://www.asncpns.com/2014/05/penerimaan-cpns-akhir-juni-ujian-cat.html Apabila pelamar memberikan keterangan/data yang tidak benar dan di kemudian hari diketahui, baik pada setiap tahapan seleksi maupun setelah diangkat menjadi CPNS/PNS, maka Kementerian atau instansi yang bersangkutan berhak menggugurkan kelulusan tersebut dan/atau memberhentikannya sebagai CPNS/PNS, menuntut ganti rugi atas kerugian negara yang terjadi akibat keterangan yang tidak benar tersebut, dan melaporkan sebagai tindak pidana ke pihak yang berwajib karena telah memberikan keterangan palsu. Informasi lebih lanjut mengenai hal-hal lain yang berkaitan dengan pengadaan CPNS tahun 2014 (pengumuman pengadaan, pelaksanaan ujian, wawancara, pengumuman kelulusan, dll) para calon pelamar disarankan untuk terus memonitor perkembangannya pada website ASNCPNS.COM Sumber : http://www.asncpns.com/2014/05/persyaratan-umum-dan-khusus-cpns.html About these ads

THE PHASES OF DRUG METABOLISM

DRUG METABOLISM - Frank J. Gonzalez and Robert H. Tukey HOW HUMANS COPE WITH EXPOSURE TO XENOBIOTICS The ability of humans to metabolize and clear drugs is a natural process that involves the same enzymatic pathways and transport systems that are utilized for normal metabolism of dietary constituents. Humans come into contact with scores of foreign chemicals or xenobiotics (substances foreign to the body) through exposure to environmental contaminants as well as in our diets. Fortunately, humans have developed a means to rapidly eliminate xenobiotics so they do not cause harm. In fact, one of the most common sources of xenobiotics in the diet is from plants that have many structurally diverse chemicals, some of which are associated with pigment production and others that are actually toxins (called phytoallexins) that protect plants against predators. A common example is poisonous mushrooms that have many toxins that are lethal to mammals, including amanitin, gyromitrin, orellanine, muscarine, ibotenic acid, muscimol, psilocybin, and coprine. Animals must be able to metabolize and eliminate such chemicals in order to consume vegetation. While humans can now choose their dietary source, a typical animal does not have this luxury and as a result is subject to its environment and the vegetation that exists in that environment. Thus, the ability to metabolize unusual chemicals in plants and other food sources is critical for survival. Drugs are considered xenobiotics and most are extensively metabolized in humans. It is worth noting that many drugs are derived from chemicals found in plants, some of which had been used in Chinese herbal medicines for thousands of years. Of the prescription drugs in use today for cancer treatment, many derive from plant species (see Chapter 51); investigating folklore claims led to the discovery of most of these drugs. It is therefore not surprising that animals utilize a means for disposing of human-made drugs that mimics the disposition of chemicals found in the diet. This capacity to metabolize xenobiotics, while mostly beneficial, has made development of drugs very time consuming and costly due in large part to (1) interindividual variations in the capacity of humans to metabolize drugs, (2) drug-drug interactions, and (3) species differences in expression of enzymes that metabolize drugs. The latter limits the use of animal models in drug development. A large number of diverse enzymes have evolved in animals that apparently only function to metabolize foreign chemicals. As will be discussed below, there are such large differences among species in the ability to metabolize xenobiotics that animal models cannot be relied upon to predict how humans will metabolize a drug. Enzymes that metabolize xenobiotics have historically been called drug-metabolizing enzymes, although they are involved in the metabolism of many foreign chemicals to which humans are exposed. Dietary differences among species during the course of evolution could account for the marked species variation in the complexity of the drug-metabolizing enzymes. Today, most xenobiotics to which humans are exposed come from sources that include environmental pollution, food additives, cosmetic products, agrochemicals, processed foods, and drugs. In general, these are lipophilic chemicals, that in the absence of metabolism would not be efficiently eliminated, and thus would accumulate in the body, resulting in toxicity. With very few exceptions, all xenobiotics are subjected to one or multiple pathways that constitute the phase 1 and phase 2 enzymatic systems. As a general paradigm, metabolism serves to convert these hydrophobic chemicals into derivatives that can easily be eliminated through the urine or the bile. In order to be accessible to cells and reach their sites of action, drugs generally must possess physical properties that allow them to move down a concentration gradient into the cell. Thus, most drugs are hydrophobic, a property that allows entry through the lipid bilayers into cells where drugs interact with their target receptors or proteins. Entry into cells is facilitated by a large number of transporters on the plasma membrane (see Chapter 2). This property of hydrophobicity would render drugs difficult to eliminate, since in the absence of metabolism, they would accumulate in fat and cellular phospholipid bilayers in cells. The xenobiotic-metabolizing enzymes convert drugs and xenobiotics into compounds that are hydrophilic derivatives that are more easily eliminated through excretion into the aqueous compartments of the tissues. Thus, the process of drug metabolism that leads to elimination plays a major role in diminishing the biological activity of a drug. For example, (S)-phenytoin, an anticonvulsant used in the treatment of epilepsy, is virtually insoluble in water. Metabolism by the phase 1 cytochrome P450 isoenzymes (CYPs) followed by phase 2 uridine diphosphate-glucuronosyltransferase (UGT) enzymes produces a metabolite that is highly water soluble and readily eliminated from the body (Figure 3-1). Metabolism also terminates the biological activity of the drug. In the case of phenytoin, metabolism also increases the molecular weight of the compound, which allows it to be eliminated more efficiently in the bile. While xenobiotic-metabolizing enzymes are responsible for facilitating the elimination of chemicals from the body, paradoxically these same enzymes can also convert certain chemicals to highly reactive toxic and carcinogenic metabolites. This occurs when an unstable intermediate is formed that has reactivity toward other compounds found in the cell. Chemicals that can be converted by xenobiotic metabolism to cancer-causing derivatives are called carcinogens. Depending on the structure of the chemical substrate, xenobiotic-metabolizing enzymes produce electrophilic metabolites that can react with nucleophilic cellular macromolecules such as DNA, RNA, and protein. This can cause cell death and organ toxicity. Reaction of these electrophiles with DNA can sometimes result in cancer through the mutation of genes such as oncogenes or tumor suppressor genes. It is generally believed that most human cancers are due to exposure to chemical carcinogens. This potential for carcinogenic activity makes testing the safety of drug candidates of vital importance. Testing for potential cancer-causing activity is particularly critical for drugs that will be used for the treatment of chronic diseases. Since each species has evolved a unique combination of xenobiotic-metabolizing enzymes, nonprimate rodent models cannot be solely used during drug development for testing the safety of new drug candidates targeted for human diseases. Nevertheless, testing in rodent models such as mice and rats can usually identify potential carcinogens. THE PHASES OF DRUG METABOLISM Xenobiotic metabolizing enzymes have historically been grouped into the phase 1 reactions, in which enzymes carry out oxidation, reduction, or hydrolytic reactions, and the phase 2 reactions, in which enzymes form a conjugate of the substrate (the phase 1 product) (Table 3-1). The phase 1 enzymes lead to the introduction of what are called functional groups, resulting in a modification of the drug, such that it now carries an -OH, -COOH, -SH, -O- or NH2 group. The addition of functional groups does little to increase the water solubility of the drug, but can dramatically alter the biological properties of the drug. Phase 1 metabolism is classified as the functionalization phase of drug metabolism; reactions carried out by phase 1 enzymes usually lead to the inactivation of an active drug. In certain instances, metabolism, usually the hydrolysis of an ester or amide linkage, results in bioactivation of a drug. Inactive drugs that undergo metabolism to an active drug are called prodrugs. An example is the antitumor drug cyclophosphamide, which is bioactivated to a cell-killing electrophilic derivative (see Chapter 51). Phase 2 enzymes facilitate the elimination of drugs and the inactivation of electrophilic and potentially toxic metabolites produced by oxidation. While many phase 1 reactions result in the biological inactivation of the drug, phase 2 reactions produce a metabolite with improved water solubility and increased molecular weight, which serves to facilitate the elimination of the drug from the tissue. Superfamilies of evolutionarily related enzymes and receptors are common in the mammalian genome; the enzyme systems responsible for drug metabolism are good examples. The phase 1 oxidation reactions are carried out by CYPs, flavin-containing monooxygenases (FMO), and epoxide hydrolases (EH). The CYPs and FMOs are composed of superfamilies of enzymes. Each superfamily contains multiple genes. The phase 2 enzymes include several superfamilies of conjugating enzymes. Among the more important are the glutathione-S-transferases (GST), UDP-glucuronosyltransferases (UGT), sulfotransferases (SULT), N-acetyltransferases (NAT), and methyltransferases (MT). These conjugation reactions usually require the substrate to have oxygen (hydroxyl or epoxide groups), nitrogen, and sulfur atoms that serve as acceptor sites for a hydrophilic moiety, such as glutathione, glucuronic acid, sulfate, or an acetyl group, that is covalently conjugated to an acceptor site on the molecule. The example of phase 1 and phase 2 metabolism of phenytoin is shown in Figure 3-1. The oxidation by phase 1 enzymes either adds or exposes a functional group, permitting the products of phase 1 metabolism to serve as substrates for the phase 2 conjugating or synthetic enzymes. In the case of the UGTs, glucuronic acid is delivered to the functional group, forming a glucuronide metabolite that is now more water soluble with a higher molecular weight that is targeted for excretion either in the urine or bile. When the substrate is a drug, these reactions usually convert the original drug to a form that is not able to bind to its target receptor, thus attenuating the biological response to the drug.

BLOOD-BRAIN BARRIER AND BLOOD-CSF BARRIER

BLOOD-BRAIN BARRIER AND BLOOD-CSF BARRIER Drugs acting in the CNS have to cross the BBB or blood-CSF barrier. These two barriers are formed by brain capillary endothelial cells and epithelial cells of the choroid plexus, respectively. Recent studies have shown that this is not only a static anatomical barrier but also a dynamic one in which efflux transporters play a role (Begley and Brightman, 2003; Sun et al., 2003). P-glycoprotein was identified initially as an efflux transporter, and it extrudes its substrate drugs on the luminal membrane of the brain capillary endothelial cells into the blood. Thus, recognition by P-glycoprotein as a substrate is a major disadvantage for drugs used to treat CNS diseases. In addition to P-glycoprotein, there is accumulating evidence for the presence of efflux transport systems for anionic drugs. The transporters involved in the efflux transport of organic anions from the CNS are being identified in the BBB and the blood-CSF barrier and include the members of organic anion transporting polypeptide (OATP1A4 and OATP1A5) and organic anion transporter (OAT3) families (Kikuchi et al., 2004; Mori et al., 2003). They facilitate the uptake process of organic compounds such as b-lactam antibiotics, statins, p-aminohippurate, H2 antagonists, and bile acids on the plasma membrane facing the brain-CSF. The transporters involved in the efflux on the membranes that face the blood still remain to be identified, although several candidate primary active transporters, such as MRP and BCRP, already have been proposed. Members of the organic anion transporting polypeptide family also mediate uptake from the blood on the plasma membrane facing blood. Further clarification of influx and efflux transporters in the barriers will enable delivery of CNS drugs efficiently into the brain while avoiding undesirable CNS side effects and help to define the mechanisms of drug-drug interactions and interindividual differences in the therapeutic CNS effects.

VECTORIAL TRANSPORT

VECTORIAL TRANSPORT The SLC type of transporter mediates either drug uptake or efflux, whereas ABC transporters mediate only unidirectional efflux. Asymmetrical transport across a monolayer of polarized cells, such as the epithelial and endothelial cells of brain capillaries, is called vectorial transport (Figure 2-5). Vectorial transport is important in the efficient transfer of solutes across epithelial or endothelial barriers. For example, vectorial transport is important for the absorption of nutrients and bile acids in the intestine. From the viewpoint of drug absorption and disposition, vectorial transport plays a major role in hepatobiliary and urinary excretion of drugs from the blood to the lumen and in the intestinal absorption of drugs. In addition, efflux of drugs from the brain via brain endothelial cells and brain choroid plexus epithelial cells involves vectorial transport. For lipophilic compounds that have sufficient membrane permeability, ABC transporters alone are able to achieve vectorial transport by extruding their substrates to the outside of cells without the help of influx transporters (Horio et al., 1990). For relatively hydrophilic organic anions and cations, coordinated uptake and efflux transporters in the polarized plasma membranes are necessary to achieve the vectorial movement of solutes across an epithelium. Common substrates of coordinated transporters are transferred efficiently across the epithelial barrier (Sasaki et al., 2002). In the liver, a number of transporters with different substrate specificities are localized on the sinusoidal membrane (facing blood). These transporters are involved in the uptake of bile acids, amphipathic organic anions, and hydrophilic organic cations into the hepatocytes. Similarly, ABC transporters on the canalicular membrane (facing bile) export such compounds into the bile. Overlapping substrate specificities between the uptake transporters (OATP family) and efflux transporters (MRP family) make the vectorial transport of organic anions highly efficient. Similar transport systems also are present in the intestine, renal tubules, and endothelial cells of the brain capillaries (Figure 2-5). Regulation of Transporter Expression. Transporter expression can be regulated transcriptionally in response to drug treatment and pathophysiological conditions, resulting in induction or down-regulation of transporter mRNAs. Recent studies have described important roles of type II nuclear receptors, which form heterodimers with the 9-cis-retinoic acid receptor (RXR), in regulating drug-metabolizing enzymes and transporters (Kullak-Ublick et al., 2004; Wang and LeCluyse, 2003). Such receptors include pregnane X receptor (PXR/NR1I2), constitutive androstane receptor (CAR/NR1I3), farnesoid X receptor (FXR/NR1H4), PPARa (peroxisome proliferator-activated receptor a), and retinoic acid receptor (RAR). Except for CAR, these are ligand-activated nuclear receptors that, as heterodimers with RXR, bind specific elements in the enhancer regions of target genes. CAR has constitutive transcriptional activity that is antagonized by inverse agonists such as androstenol and androstanol and induced by barbiturates. PXR, also referred to as steroid X receptor (SXR) in humans, is activated by synthetic and endogenous steroids, bile acids, and drugs such as clotrimazole, phenobarbital, rifampicin, sulfinpyrazone, ritonavir, carbamazepine, phenytoin, sulfadimidine, taxol, and hyperforin (a constituent of St. John's wort). Table 2-1 summarizes the effects of drug activation of type II nuclear receptors on expression of transporters. The potency of activators of PXR varies among species such that rodents are not necessarily a model for effects in humans. There is an overlap of substrates between CYP3A4 and P-glycoprotein, and PXR mediates coinduction of CYP3A4 and P-glycoprotein, supporting their synergetic cooperation in efficient detoxification. See Table 3-4 and Figure 3-13 for information on the role of type II nuclear receptors in induction of drug-metabolizing enzymes. MOLECULAR STRUCTURES OF TRANSPORTERS Predictions of secondary structure of membrane transport proteins based on hydropathy analysis indicate that membrane transporters in the SLC and ABC superfamilies are multi-membrane-spanning proteins. A typical predicted secondary structure of the ABC transporter MRP2 (ABCC2) is shown in Figure 2-6. However, understanding the secondary structure of a membrane transporter provides little information on how the transporter functions to translocate its substrates. For this, information on the tertiary structure of the transporter is needed, along with complementary molecular information about the residues in the transporter that are involved in the recognition, association, and dissociation of its substrates. To obtain high-resolution structures of membrane proteins, the proteins first must be crystallized, and then the crystal structure must be deduced from analysis of x-ray diffraction patterns. Crystal structures generally are difficult to obtain for membrane proteins primarily because of their amphipathic needs for stabilization. Further, membrane proteins generally are in low abundance, so obtaining sufficient quantities for structural determination is difficult. The few membrane transporters that have been crystallized are bacterial proteins that can be expressed in high abundance. Information on two representative membrane transporters that have been crystallized and analyzed at relatively high resolution (<4 A) serves to illustrate some basic structural properties of membrane transporters. One of the transporters, MsbA, is an ABC transporter from E. coli with homology to multidrug-resistance efflux pumps in mammals. The second transporter, LacY, is a proton symporter, also from E. coli, that translocates lactose and other oligosaccharides. Each of these transporters is illustrative of a different transport mechanism. Lipid Flippase (MsbA). MsbA is an ABC transporter in E. coli that, like other ABC transporters, hydrolyzes ATP to export its substrate. Based on an x-ray crystal structure, MsbA forms a homodimer consisting of two six-transmembrane units, each with a nucleotide-binding domain on the cytoplasmic surface (Chang and Roth, 2001) (Figure 2-7). The hexaspanning unit consists of six a-helices. There is a central chamber with an asymmetrical distribution of charged residues. A transport mechanism that is consistent with this asymmetrical distribution of charges is a "flippase" mechanism. That is, substrates in the inner leaflet of the bilayer are recognized by MsbA and then flipped to the outer leaflet of the bilayer. This hypothetical mechanism, although intriguing, leaves many questions unanswered. For example, how is the energy of ATP hydrolysis coupled to the flipping process? Once in the outer leaflet, how are substrates translocated to the extracellular space? Nevertheless, from this structure and other structures, we now know that transmembrane domains form a-helices, that six-unit dimers are central to the transport mechanism, and that there is an asymmetrical distribution of charged residues in a central chamber. Lactose Permease Symporter (LacY). Lactose permease is a bacterial transporter that belongs to the major facilitator superfamily (MFS). This transporter is a proton-coupled symporter. A high-resolution X-ray crystal structure has been obtained for the protonated form of a mutant of LacY (C154G) at a 3.5-Aa-resolution (Abramson et al., 2003) (Figure 2-8). In brief, LacY is comprised of two units of six membrane-spanning a-helices. The crystal structure showed substrate located at the interface of the two units and in the middle of the membrane. This location is consistent with an alternating-access transport mechanism in which the substrate recognition site is accessible to the cytosolic and then the extracellular surface but not to both simultaneously. Eight helices form the surface of the hydrophilic cavity, and each contains proline and glycine residues that result in kinks in the cavity. From LacY, we now know that as in the case of MsbA, six membrane-spanning a-helices are critical structural units for transport by LacY. TRANSPORTER SUPERFAMILIES IN THE HUMAN GENOME Introduction Two major gene superfamilies play critical roles in the transport of drugs across plasma and other biological membranes: the SLC and ABC superfamilies. Web sites that have information on these families include http://nutrigene.4t.com/humanabc.htm (ABC superfamily), http://www.bioparadigms.org/slc/intro.asp (SLC superfamily), http://www.pharmaconference.org/slctable.asp (SLC superfamily), and http://www.TP_Search.jp/ (drug transporters). Information on pharmacogenetics of these transporters can be found in Chapter 4 and at http://www.pharmgkb.org and http://www.pharmacogenetics.ucsf.edu. Slc Transporters. The solute carrier (SLC) superfamily includes 43 families and represents approximately 300 genes in the human genome. The nomenclature of the transporters within each family is listed under the Human Genome Organization (HUGO) Nomenclature Committee database at http://www.gene.ucl.ac.uk/nomenclature/. Table 2-2 lists the families in the human SLC superfamily and some of the genetic diseases that are associated with members of selected families. The family name provides a description of the function(s) of each family. However, some caution should be exercised in interpretation of family names because individual family members may have vastly different specificities or functional roles. All the SLC families with members in the human genome were reviewed recently (Hediger, 2004). In brief, transporters in the SLC superfamily transport diverse ionic and nonionic endogenous compounds and xenobiotics. SLC superfamily transporters may be facilitated transporters or secondary active symporters or antiporters. The first SLC family transporter was cloned in 1987 by expression cloning in Xenopus laevis oocytes (Hediger et al., 1987). Since then, many transporters in the SLC superfamily have been cloned and characterized functionally. Predictive models defining important characteristics of substrate binding and knockout mouse models defining the in vivo role of specific transporters have been constructed for many SLC transporters (Chang et al., 2004; Ocheltree et al., 2004). In general, in this chapter we focus on SLC transporters in the human genome, which are designated by capital letters (SLC transporters in rodent genomes are designated by lowercase letters). ABC Superfamily. In 1976, Juliano and Ling reported that overexpression of a membrane protein in colchicine -resistant Chinese hamster ovary cells also resulted in acquired resistance to many structurally unrelated drugs (i.e., multidrug resistance) (Juliano and Ling, 1976). Since the cDNA cloning of this first mammalian ABC protein (P-glycoprotein/MDR1/ABCB1), the ABC superfamily has continued to grow; it now consists of 49 genes, each containing one or two conserved ABC regions (Borst and Elferink, 2002). The ABC region is a core catalytic domain of ATP hydrolysis and contains Walker A and B sequences and an ABC transporter-specific signature C sequence (Figure 2-6). The ABC regions of these proteins bind and hydrolyze ATP, and the proteins use the energy for uphill transport of their substrates across the membrane. Although some ABC superfamily transporters contain only a single ABC motif, they form homodimers (BCRP/ABCG2) or heterodimers (ABCG5 and ABCG8) that exhibit a transport function. ABC transporters (e.g., MsbA) (Figure 2-7) also are found in prokaryotes, where they are involved predominantly in the import of essential compounds that cannot be obtained by passive diffusion (sugars, vitamins, metals, etc.). By contrast, most ABC genes in eukaryotes transport compounds from the cytoplasm to the outside or into an intracellular compartment (endoplasmic reticulum, mitochondria, peroxisomes). ABC transporters can be divided into seven groups based on their sequence homology: ABCA (12 members), ABCB (11 members), ABCC (13 members), ABCD (4 members), ABCE (1 member), ABCF (3 members), and ABCG (5 members). ABC genes are essential for many cellular processes, and mutations in at least 13 of these genes cause or contribute to human genetic disorders (Table 2-3). In addition to conferring multidrug resistance (Sadee et al., 1995), an important pharmacological aspect of these transporters is xenobiotic export from healthy tissues. In particular, MDR1/ABCB1, MRP2/ABCC2, and BCRP/ABCG2 have been shown to be involved in overall drug disposition (Leslie et al., 2005). Properties of ABC Transporters Related to Drug Action The tissue distribution of drug-related ABC transporters in the body is summarized in Table 2-4 together with information about typical substrates. Tissue Distribution of Drug-Related ABC Transporters. MDR1 (ABCB1), MRP2 (ABCC2), and BCRP (ABCG2) are all expressed in the apical side of the intestinal epithelia, where they serve to pump out xenobiotics, including many clinically relevant drugs. The kidney and liver are major organs for overall systemic drug elimination from the body. The liver also plays a role in presystemic drug elimination. Key to the vectorial excretion of drugs into urine or bile, ABC transporters are expressed in the polarized tissues of kidney and liver: MDR1, MRP2, and MRP4 (ABCC4) on the brush-border membrane of renal epithelia, and MDR1, MRP2, and BCRP on the bile canalicular membrane of hepatocytes. Some ABC transporters are expressed specifically on the blood side of the endothelial or epithelial cells that form barriers to the free entrance of toxic compounds into naive tissues: the BBB (MDR1 and MRP4 on the luminal side of brain capillary endothelial cells), the blood-cerebrospinal fluid (CSF) barrier (MRP1 and MRP4 on the basolateral blood side of choroid plexus epithelia), the blood-testis barrier (MRP1 on the basolateral membrane of mouse Sertoli cells and MDR1 in several types of human testicular cells), and the blood-placenta barrier (MDR1, MRP2, and BCRP on the luminal maternal side and MRP1 on the antiluminal fetal side of placental trophoblasts). Substrate Specificity of ABC Transporters. MDR1/ABCB1 substrates tend to share a hydrophobic planar structure with positively charged or neutral moieties as described in Table 2-4 (see also Ambudkar et al., 1999). These include structurally and pharmacologically unrelated compounds, many of which are also substrates of CYP3A4, a major drug-metabolizing enzyme in the human liver and GI tract. Such overlapping substrate specificity implies a synergistic role for MDR1 and CYP3A4 in protecting the body by reducing the intestinal absorption of xenobiotics (Zhang and Benet, 2001). After being taken up by enterocytes, some drug molecules are metabolized by CYP3A4. Drug molecules that escape metabolic conversion are eliminated from the cells via MDR1 and then reenter the enterocytes. The intestinal residence time of the drug is prolonged with the aid of MDR1, thereby increasing the chance of local metabolic conversion by the CYP3A4 (see Chapter 3). MRP/ABCC Family. The substrates of transporters in the MRP/ABCC family are mostly organic anions. The substrate specificities of MRP1 and MRP2 are similar: Both accept glutathione and glucuronide conjugates, sulfated conjugates of bile salts, and nonconjugated organic anions of an amphipathic nature (at least one negative charge and some degree of hydrophobicity). They also transport neutral or cationic anticancer drugs, such as vinca alkaloids and anthracyclines, possibly via a cotransport or symport mechanism with reduced glutathione (GSH). MRP3 also has a substrate specificity that is similar to that of MRP2 but with a lower transport affinity for glutathione conjugates compared with MRP1 and MRP2. Most characteristic MRP3 substrates are monovalent bile salts, which are never transported by MRP1 and MRP2. Because MRP3 is expressed on the sinusoidal side of hepatocytes and is induced under cholestatic conditions, backflux of toxic bile salts and bilirubin glucuronides into the blood circulation is considered to be its physiological function. MRP4 and MRP5 have narrower substrate specificities. They accept nucleotide analogues and clinically important anti-human immunodeficiency virus (HIV) drugs. Although some transport substrates have been identified for MRP6, no physiologically important endogenous substrates have been identified that explain the mechanism of the MRP6-associated disease pseudoxanthoma. BCRP/ABCG2. BCRP accepts both neutral and negatively charged molecules, including cytotoxic compounds (e.g., mitoxantrone, topotecan, flavopiridol, and methotrexate), sulfated conjugates of therapeutic drugs and hormones (e.g., estrogen sulfate), and toxic compounds found in normal food [2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and pheophorbide A, a chlorophyll catabolite]. Physiological Roles of ABC Transporters. The physiological significance of the ABC transporters is illustrated by studies involving knockout animals or patients with genetic defects in these transporters. Mice deficient in MDR1 function are viable and fertile and do not display obvious phenotypic abnormalities other than hypersensitivity to toxic drugs, including the neurotoxic pesticide ivermectin (one hundredfold) and the carcinostatic drug vinblastine (threefold) (Schinkel et al., 1994). mrp1 (-/-) mice are also viable and fertile without any obvious difference in litter size. However, these mice are hypersensitive to the anticancer drug etoposide. Damage is especially severe in the testis, kidney, and oropharyngeal mucosa, where MRP1 is expressed on the basolateral membrane. Moreover, these mice have an impaired response to an arachidonic acid-induced inflammatory stimulus, which is likely due to a reduced secretion of leukotriene C4 from mast cells, macrophages, and granulocytes. MRP2-deficient rats (TR- and EHBR) and Dubin-Johnson syndrome patients are normal in appearance except for mild jaundice owing to impaired biliary excretion of bilirubin glucuronide (Ito et al., 1997; Paulusma et al., 1996). BCRP knockout mice are viable but highly sensitive to the dietary chlorophyll catabolite phenophorbide, which induces phototoxicity. These mice also exhibit protoporphyria, with a tenfold increase in protoporphyrin IX accumulation in erythrocytes, resulting in photosensitivity. This protoporphyria is caused by the impaired function of BCRP in bone marrow: Knockout mice transplanted with bone marrow from wild-type mice become normal with respect to protoporphyrin IX level in the erythrocytes and photosensitivity. As described earlier, complete absence of these drug-related ABC transporters is not lethal and even can remain unrecognized without exogenous perturbation owing to food, drugs, or toxins. Inhibition of physiologically important ABC transporters (especially those related directly to the genetic diseases described in Table 2-3) by drugs should be avoided to reduce the incidence of drug-induced side effects. ABC Transporters in Drug Absorption and Elimination. With respect to clinical medicine, MDR1 is the most important ABC transporter yet identified, and digoxin is one of the most widely studied of its substrates. The systemic exposure to orally administered digoxin (as assessed by the area under the plasma digoxin concentration-time curve) is increased by coadministration of rifampin (an MDR1 inducer) and is negatively correlated with the MDR1 protein expression in the human intestine. MDR1 is also expressed on the brush-border membrane of renal epithelia, and its function can be monitored using digoxin as a probe drug. Digoxin undergoes very little degradation in the liver, and renal excretion is the major elimination pathway (>70%) in humans. Several studies in healthy subjects have been performed with MDR1 inhibitors (e.g., quinidine, verapamil, vaspodar, spironolactone, clarythromycin, and ritonavir) with digoxin as a probe drug, and all resulted in a marked reduction in the renal excretion of digoxin. Similarly, the intestinal absorption of cyclosporine is also related mainly to the MDR1 level rather than to the CYP3A4 level, although cyclosporine is a substrate of both CYP3A4 and MDR1. Alteration of MDR1 activity by inhibitors (drug-drug interactions) affects oral absorption and renal clearance. Drugs with narrow therapeutic windows (such as the cardiac glycoside digoxin and the immunosuppressants cyclosporine and tacrolimus) should be used with great care if MDR1-based drug-drug interactions are likely. Despite the broad substrate specificity and distinct localization of MRP2 and BCRP in drug-handling tissues (both expressed on the canalicular membrane of hepatocytes and the brush-border membrane of enterocytes), there has been very little integration of clinically relevant information. Part of the problem lies in distinguishing the biliary transport activities of MRP2 and BCRP from the contribution of the hepatic uptake transporters of the OATP family. Most MRP2 or BCRP substrates also can be transported by the OATP family transporters on the sinusoidal membrane. The rate-limiting process for systemic elimination is uptake in most cases. Under such conditions, the effect of drug-drug interactions (or genetic variants) in these biliary transporters may be difficult to identify. Despite such practical difficulties, there is a steady increase in the information about genetic variants and their effects on transporter expression and activity in vitro. Variants of BCRP with high allele frequencies (0.184 for V12M and 0.239 for Q141K) have been found to alter the substrate specificity in cellular assays. The clinical impact of these variants and drug-drug interactions needs to be studied in more detail in humans and under in vivo conditions using appropriate probe drugs. GENETIC VARIATION IN MEMBRANE TRANSPORTERS: IMPLICATIONS FOR CLINICAL DRUG RESPONSE Inherited defects in membrane transport have been known for many years, and the genes associated with several inherited disorders of membrane transport have been identified [Table 2-2 (SLC) and Table 2-3 (ABC)]. Reports of polymorphisms in membrane transporters that play a role in drug response have appeared only recently, but the field is growing rapidly. Cellular studies have focused on genetic variation in only a few drug transporters, but progress has been made in characterizing the functional impact of variants in these transporters. Further, large-scale studies in the area of single-nucleotide polymorphisms (SNPs) in membrane transporters and cellular characterization of transporter variants have been performed (Burman et al., 2004; Gray et al., 2004; Leabman et al., 2003; Osato et al., 2003; Shu et al., 2003) (see Chapter 4). The clinical impact of membrane transporter variants on drug response has been studied only recently. Like the cellular studies, the clinical studies have focused on a limited number of transporters. The most widely studied drug transporter is P-glycoprotein (MDR1, ABCB1), and results from clinical studies have been controversial. Associations of the ABCB1 genotype with responses to anticancer drugs, antiviral agents, immunosuppressants, antihistamines, cardiac glycosides, and anticonvulsants have been described (Anglicheau et al., 2003; Drescher et al., 2002; Fellay et al., 2002; Hoffmeyer et al., 2000; Illmer et al., 2002; Johne et al., 2002; Macphee et al., 2002; Pauli-Magnus et al., 2003; Sai et al., 2003; Sakaeda et al., 2003; Siddiqui et al., 2003; Verstuyft et al., 2003). ABCB1 SNPs also have been associated with tacrolimus and nortriptyline neurotoxicity (Roberts et al., 2002; Yamauchi et al., 2002) and susceptibility for developing ulcerative colitis, renal cell carcinoma, and Parkinson's disease (Drozdzik et al., 2003; Schwab et al., 2003; Siegsmund et al., 2002). Recently, two common SNPs in SLCO1B1 (OATP1B1) have been associated with elevated plasma levels of pravastatin, a widely used drug for the treatment of hypercholesterolemia (Mwinyi et al., 2004; Niemi et al., 2004) (see Chapter 35). TRANSPORTERS INVOLVED IN PHARMACOKINETICS Hepatic Transporters Drug transporters play an important role in pharmacokinetics (Koepsell, 1998; Zamek-Gliszczynski and Brouwer, 2004) (Figure 2-1). Hepatic uptake of organic anions (e.g., drugs, leukotrienes, and bilirubin), cations, and bile salts is mediated by SLC-type transporters in the basolateral (sinusoidal) membrane of hepatocytes: OATPs (SLCO) (Abe et al., 1999; Konig et al., 2000) and OATs (SLC22) (Sekine et al., 1998), OCTs (SLC22) (Koepsell, 1998) and NTCP (SLC10A1) (Hagenbuch et al., 1991), respectively. These transporters mediate uptake by either facilitated or secondary active mechanisms. ABC transporters such as MRP2, MDR1, BCRP, BSEP, and MDR2 in the bile canalicular membrane of hepatocytes mediate the efflux (excretion) of drugs and their metabolites, bile salts, and phospholipids against a steep concentration gradient from liver to bile. This primary active transport is driven by ATP hydrolysis. Some ABC transporters are also present in the basolateral membrane of hepatocytes and may play a role in the efflux of drugs back into the blood, although their physiological role remains to be elucidated. Drug uptake followed by metabolism and excretion in the liver is a major determinant of the systemic clearance of many drugs. Since clearance ultimately determines systemic blood levels, transporters in the liver play key roles in setting drug levels. Vectorial transport of drugs from the circulating blood to the bile using an uptake transporter (OATP family) and an efflux transporter (MRP2) is important for determining drug exposure in the circulating blood and liver. Moreover, there are many other uptake and efflux transporters in the liver (Figure 2-9). Two examples illustrate the importance of vectorial transport in determining drug exposure in the circulating blood and liver: HMG-CoA reductase inhibitors and angiotensin-converting enzyme (ACE) inhibitors. HMG-CoA Reductase Inhibitors. Statins are cholesterol-lowering agents that reversibly inhibit HMG-CoA reductase, which catalyzes a rate-limiting step in cholesterol biosynthesis (see Chapter 35). Statins affect serum cholesterol by inhibiting cholesterol biosynthesis in the liver, and this organ is their main target. On the other hand, exposure of extrahepatic cells in smooth muscle to these drugs may cause adverse effects. Among the statins, pravastatin, fluvastatin, cerivastatin, atorvastatin, rosuvastatin, andpitavastatin are given in a biologically active open-acid form, whereas simvastatin and lovastatin are administered as inactive prodrugs with lactone rings. The open-acid statins are relatively hydrophilic and have low membrane permeabilities. However, most of the statins in the acid form are substrates of uptake transporters, so they are taken up efficiently by the liver and undergo enterohepatic circulation (Figures 2-5 and 2-9). In this process, hepatic uptake transporters such as OATP1B1 and efflux transporters such as MRP2 act cooperatively to produce vectorial transcellular transport of bisubstrates in the liver. The efficient first-pass hepatic uptake of these statins by OATP1B1 after their oral administration helps to exert the pharmacological effect and also minimizes the escape of drug molecules into the circulating blood, thereby minimizing the exposure in a target of adverse response, smooth muscle. Recent studies indicate that the genetic polymorphism of OATP1B1 also affects the function of this transporter (Tirona et al., 2001). Temocapril. Temocapril is an ACE inhibitor (see Chapter 30). Its active metabolite, temocaprilat, is excreted both in the bile and in the urine via the liver and kidney, respectively, whereas other ACE inhibitors are excreted mainly via the kidney. The special feature of temocapril among ACE inhibitors is that the plasma concentration of temocaprilat remains relatively unchanged even in patients with renal failure. However, the plasma area under the curve AUC of enalaprilat and other ACE inhibitors is markedly increased in patients with renal disorders. Temocaprilat is a bisubstrate of the OATP family and MRP2, whereas other ACE inhibitors are not good substrates of MRP2 (although they are taken up into the liver by the OATP family). Taking these findings into consideration, the affinity for MRP2 may dominate in determining the biliary excretion of any series of ACE inhibitors. Drugs that are excreted into both the bile and urine to the same degree thus are expected to exhibit minimum interindividual differences in their pharmacokinetics. Irinotecan (CPT-11). Irinotecan hydrochloride (CPT-11) is a potent anticancer drug, but late-onset gastrointestinal toxic effects, such as severe diarrhea, make it difficult to use CPT-11 safely. After intravenous administration, CPT-11 is converted to SN-38, an active metabolite, by carboxy esterase. SN-38 is subsequently conjugated with glucuronic acid in the liver. SN-38 and SN-38 glucuronide are then excreted into the bile by MRP2. Some studies have shown that the inhibition of MRP2-mediated biliary excretion of SN-38 and its glucuronide by coadministration of probenecid reduces the drug-induced diarrhea, at least in rats. For additional details, see Figures 3-5 and 3-7. Drug-Drug Interactions Involving Transporter-Mediated Hepatic Uptake. Since drug transporters are determinants of the elimination rate of drugs from the body, transporter-mediated hepatic uptake can be the cause of drug-drug interactions involving drugs that are actively taken up into the liver and metabolized and/or excreted in the bile. Cerivastatin (currently withdrawn), an HMG-CoA reductase inhibitor, is taken up into the liver via transporters (especially OATP1B1) and subsequently metabolized by CYP2C8 and CYP3A4. Its plasma concentration is increased four- to fivefold when coadministered with cyclosporin A. Transport studies using cryopreserved human hepatocytes and OATP1B1-expressing cells suggest that this clinically relevant drug-drug interaction is caused by inhibition of OATP1B1-mediated hepatic uptake (Shitara et al., 2003). However, cyclosporin A inhibits the metabolism of cerivastatin only to a limited extent, suggesting a low possibility of serious drug-drug interactions involving the inhibition of metabolism. Cyclosporin A also increases the plasma concentrations of other HMG-CoA reductase inhibitors. It markedly increases the plasma AUC of pravastatin, pitavastatin, and rosuvastatin, which are minimally metabolized and eliminated from the body by transporter-mediated mechanisms. Therefore, these pharmacokinetic interactions also may be due to transporter-mediated hepatic uptake. However, the interactions of cyclosporin A with prodrug-like statins (lactone form) such as simvastatin and lovastatin are mediated by CYP3A4. Gemfibrozil is another cholesterol-lowering agent that acts by a different mechanism and also causes a severe pharmacokinetic interaction with cerivastatin. Gemfibrozil glucuronide inhibits the CYP2C8-mediated metabolism and OATP1B1-mediated uptake of cerivastatin more potently than does gemfibrozil. Laboratory data show that the glucuronide is highly concentrated in the liver versus plasma probably owing to transporter-mediated active uptake and intracellular formation of the conjugate. Therefore, it may be that gemfibrozil glucuronide, concentrated in the hepatocytes, inhibits the CYP2C8-mediated metabolism of cerivastatin. Gemfibrozil markedly (four- to fivefold) increases the plasma concentration of cerivastatin but does not greatly increase (1.3 to 2 times) that of unmetabolized statins pravastatin, pitavastatin, and rosuvastatin, a result that also suggests that this interaction is caused by inhibition of metabolism. Thus, when an inhibitor of drug-metabolizing enzymes is highly concentrated in hepatocytes by active transport, extensive inhibition of the drug-metabolizing enzymes may be observed because of the high concentration of the inhibitor in the vicinity of the drug-metabolizing enzymes. The Contribution of Specific Transporters to the Hepatic Uptake of Drugs. Estimating the contribution of transporters to the total hepatic uptake is necessary for understanding their importance in drug disposition. This estimate can help to predict the extent to which a drug-drug interaction or a genetic polymorphism of a transporter may affect drug concentrations in plasma and liver. The contribution to hepatic uptake has been estimated successfully for CYP-mediated metabolism by using neutralizing antibody and specific chemical inhibitors. Unfortunately, specific inhibitors or antibodies for important transporters have not been identified yet, although some relatively specific inhibitors have been discovered. The contribution of transporters to hepatic uptake can be estimated from in vitro studies. Injection of cRNA results in transporter expression on the plasma membrane of Xenopus laevis oocytes (Hagenbuch et al., 1996). Subsequent hybridization of the cRNA with its antisense oligonucleotide specifically reduces its expression. Comparison of the drug uptake into cRNA-injected oocytes in the presence and absence of antisense oligonucleotides clarifies the contribution of a specific transporter. Second, a method using reference compounds for specific transporters has been proposed. The reference compounds should be specific substrates for a particular transporter. The contribution of a specific transporter can be calculated from the uptake of test compounds and reference compounds into hepatocytes and transporter-expressing systems (Hirano et al., 2004): (2-13) where CLhep,ref and CLexp,ref represent the uptake of reference compounds into hepatocytes and transporter-expressing cells, respectively, and CLhep,test and CLexp,test represent the uptake of test compounds into the corresponding systems. For example, the contributions of OATP1B1 and OATP1B3 to the hepatic uptake of pitavastatin have been estimated using estrone 3-sulfate and cholecystokinine octapeptide (CCK8) as reference compounds for OATP1B1 and OATP1B3, respectively. However, for many transporters, reference compounds specific to the transporter are not available. Renal Transporters Secretion in the kidney of structurally diverse molecules including many drugs, environmental toxins and carcinogens is critical in the body's defense against foreign substances. The specificity of secretory pathways in the nephron for two distinct classes of substrates, organic anions and cations, was first described decades ago, and these pathways were well characterized using a variety of physiological techniques including isolated perfused nephrons and kidneys, micropuncture techniques, cell culture methods, and isolated renal plasma membrane vesicles. However, not until the mid-1990s were the molecular identities of the organic anion and cation transporters revealed. During the past decade, molecular studies have identified and characterized the renal transporters that play a role in drug elimination, toxicity, and response. Thus, we now can describe the overall secretory pathways for organic cations and their molecular and functional characteristics. Although the pharmacological focus is often on the kidney, there is useful information on the tissue distribution of these transporters. Molecular studies using site-directed mutagenesis have identified substrate-recognition and other functional domains of the transporters, and genetic studies of knockout mouse models have been used to characterize the physiological roles of individual transporters. Recently, studies have identified and functionally analyzed genetic polymorphisms and haplotypes of the relevant transporters in humans. Our understanding of organic anion transport has progressed in a similar fashion. In some cases, transporters that are considered organic anion or organic cation transporters have dual specificity for anions and cations. The following section summarizes recent work on human transporters and includes some information on transporters in other mammals. An excellent review of renal organic anion and cation transport has been published recently (Wright and Dantzler, 2004). Organic Cation Transport. Structurally diverse organic cations are secreted in the proximal tubule (Dresser et al., 2001; Koepsell and Endou, 2004; Wright and Dantzler, 2004). Many secreted organic cations are endogenous compounds (e.g., choline, N-methylnicotinamide, and dopamine), and renal secretion appears to be important in eliminating excess concentrations of these substances. However, a primary function of organic cation secretion is ridding the body of xenobiotics, including many positively charged drugs and their metabolites (e.g., cimetidine, ranitidine, metformin, procainamide, and N-acetylprocainamide), and toxins from the environment (e.g., nicotine). Organic cations that are secreted by the kidney may be either hydrophobic or hydrophilic. Hydrophilic organic drug cations generally have molecular weights of less than 400 daltons; a current model for their secretion in the proximal tubule of the nephron is shown in Figure 2-10. For the transepithelial flux of a compound (e.g., secretion), it is essential for the compound to traverse two membranes sequentially, the basolateral membrane facing the blood side and the apical membrane facing the tubular lumen. Distinct transporters on each membrane mediate each step of transport. Organic cations appear to cross the basolateral membrane by three distinct transporters in the SLC family 22 (SCL22): OCT1 (SLC22A1), OCT2 (SLC22A2), and OCT3 (SLC22A3). Organic cations are transported across this membrane down their electrochemical gradient (-70 mV). Previous studies in isolated basolateral membrane vesicles demonstrate the presence of a potential-sensitive mechanism for organic cations. The cloned transporters OCT1, OCT2, and OCT3 are all potential sensitive and mechanistically coincide with previous studies of isolated basolateral membrane vesicles. Transport of organic cations from cell to tubular lumen across the apical membrane occurs via an electroneutral proton-organic cation exchange mechanism in a variety of species, including human, dog, rabbit, and cat. Transporters assigned to the apical membrane are in the SLC22 family and termed novel organic cation transporters (OCTNs). In humans, these include OCNT1 (SLC22A4) and OCTN2 (SLC22A5). These bifunctional transporters are involved not only in organic cation secretion but also in carnitine reabsorption. In the reuptake mode, the transporters function as Na+ cotransporters, relying on the inwardly driven Na+ gradient created by Na+,K+-ATPase to move carnitine from tubular lumen to cell. In the secretory mode, the transporters appear to function as proton-organic cation exchangers. That is, protons move from tubular lumen to cell interior in exchange for organic cations, which move from cytosol to tubular lumen. The inwardly directed proton gradient (from tubular lumen to cytosol) is maintained by transporters in the SLC9 family (NHEs), which are Na+/H+ exchangers (antiporters). The bifunctional mechanism of OCTN1 and OCTN2 may not totally explain the organic cation-proton exchange mechanism that has been described in many studies in isolated plasma membrane vesicles. Of the two steps involved in secretory transport, transport across the luminal membrane appears to be rate-limiting. OCT1 (SLC22A1). OCT1 (SLC22A1) was first cloned from a rat cDNA library (Koepsell and Endou, 2004). Subsequently, orthologs were cloned from mouse, rabbit, and humans. Mammalian isoforms of OCT1, which vary in length from 554 to 556 amino acids, have 12 putative transmembrane domains (Figure 2-11) and include several N-linked glycosylation sites. A long extracellular loop between transmembrane domains 1 and 2 is characteristic of the OCTs. The gene for the human OCT1 is mapped to chromosome 6 (6q26). There are four splice variants in human tissues, one of which is functionally active, OCT1G/L554 (Hayer et al., 1999). In humans, OCT1 is expressed primarily in the liver, with some expression in heart, intestine, and skeletal muscle. In mouse and rat, OCT1 is also abundant in the kidney, whereas in humans, very modest levels of OCT1 mRNA transcripts are detected in kidney. The transport mechanism of OCT1 is electrogenic and saturable for transport of model small-molecular-weight organic cations including tetraethylammonium (TEA) and dopamine. Interestingly, OCT1 also can operate as an exchanger, mediating organic cation-organic cation exchange. That is, loading cells with organic cations such as unlabeled TEA can trans-stimulate the inward flux of organic cations such as MPP+. It also should be noted that organic cations can transinhibit OCT1. In particular, the hydrophobic organic cations quinine and quinidine, which are poor substrates of OCT1, when present on the cytosolic side of a membrane, can inhibit (transinhibit) influx of organic cations via OCT1. The human OCT1 generally accepts a wide array of monovalent organic cations with molecular weights of less than 400 daltons, including many drugs (e.g., procainamide, metformin, and pindolol) (Dresser et al., 2001). Species differences in the substrate specificity of OCT1 mammalian orthologs have been described. Inhibitors of OCT1 are generally more hydrophobic. Detailed structure-activity relationships have established that the pharmacophore of OCT1 consists of three hydrophobic arms and a single cationic recognition site. The kinetics of uptake and inhibition of model compounds with human OCT1 differ among studies and may be related to experimental techniques, including a range of heterologous expression systems. Key residues that contribute to the charge specificity of OCT1 have been identified by site-directed mutagenesis studies and include a highly conserved aspartate residue (corresponding to position 475 in the rat ortholog of OCT1) that appears to be part of the monoamine recognition site. Since OCT1 mammalian orthologs have greater than 80% amino acid identity, evolutionarily nonconserved residues among mammalian species clearly are involved in specificity differences (Wright and Dantzler, 2004). OCT2 (SLC22A2). OCT2 (SLC22A2) was first cloned from a rat kidney cDNA library in 1996 (Okuda et al., 1996). Human, rabbit, mouse, and pig orthologs all have been cloned. Mammalian orthologs range in length from 553 through 555 amino acids. Similar to OCT, OCT2 is predicted to have 12 transmembrane domains, including one N-linked glycosylation site. OCT2 is located adjacent to OCT1 on chromosome 6 (6q26). A single splice variant of human OCT2, termed OCT2-A, has been identified in human kidney. OCT2-A, which is a truncated form of OCT2, appears to have a lower Km (or greater affinity) for substrates than OCT2, although a lower affinity has been observed for some inhibitors (Urakami et al., 2002). Human, mouse, and rat orthologs of OCT2 are expressed in abundance in human kidney and to some extent in neuronal tissue such as choroid plexus. In the kidney, OCT2 is localized to the proximal tubule and to distal tubules and collecting ducts. In the proximal tubule, OCT2 is restricted to the basolateral membrane. OCT2 mammalian species orthologs are greater than 80% identical, whereas OCT1 and OCT2 paralogs are approximately 70% identical. The transport mechanism of OCT2 is similar to that of OCT1. In particular, OCT2-mediated transport of model organic cations MPP+ and TEA is electrogenic, but like OCT1, OCT2 can support organic cation-organic cation exchange (Koepsell et al., 2003). Some studies show modest proton-organic cation exchange. More hydrophobic organic cations may inhibit OCT2 but may not be translocated by it. Like OCT1, OCT2 generally accepts a wide array of monovalent organic cations with molecular weights of less than 400 daltons. The apparent affinities of the human OCT1 and OCT2 paralogs for some organic cation substrates and inhibitors have been shown to be different in side-by-side comparison studies. Isoform-specific inhibitors of the OCTs are needed to determine the relative importance of OCT1 and OCT2 in the renal clearance of compounds in rodents, in which both isoforms are present in kidney. OCT2 is also present in neuronal tissues. However, studies with monoamine neurotransmitters demonstrate that dopamine, serotonin, histamine, and norepinephrine have low affinities for OCT2. These studies suggest that OCT2 may play a housekeeping role in neurons, taking up only excess concentrations of neurotransmitters. OCT2 also may be involved in recycling of neurotransmitters by taking up breakdown products, which in turn enter monoamine synthetic pathways. OCT3 (SLC22A3). OCT3 (SLC22A3) was cloned initially from rat placenta (Kekuda et al., 1998). Human and mouse orthologs have also been cloned. OCT3 consists of 551 amino acids and is predicted to have 12 transmembrane domains, including three N-linked glycosylation sites. hOCT3 is located in tandem with OCT1 and OCT2 on chromosome 6. Tissue distribution studies suggest that human OCT3 is expressed in liver, kidney, intestine, and placenta, although it appears to be expressed in considerably less abundance than OCT2 in the kidney. Like OCT1 and OCT2, OCT3 appears to support electrogenic potential-sensitive organic cation transport. Although the specificity of OCT3 is similar to that of OCT1 and OCT2, it appears to have quantitative differences in its affinities for many organic cations. Some studies have suggested that OCT3 is the extraneuronal monoamine transporter based on its substrate specificity and potency of interaction with monoamine neurotransmitters. Because of its relatively low abundance in the kidney, OCT3 may play only a limited role in renal drug elimination. OCTN1 (SLC22A4). OCTN1, cloned originally from human fetal liver, is expressed in the adult kidney, trachea, and bone marrow (Tamai et al., 1997). The functional characteristics of OCTN1 suggest that it operates as an organic cation-proton exchanger. OCTN1-mediated influx of model organic cations is enhanced at alkaline pH, whereas efflux is increased by an inwardly directed proton gradient. OCTN1 contains a nucleotide-binding sequence motif, and transport of its substrates appears to be stimulated by cellular ATP content. OCTN1 also can function as an organic cation-organic cation exchanger. Although the subcellular localization of OCTN1 has not been demonstrated clearly, available data collectively suggest that OCTN1 functions as a bidirectional pH- and ATP-dependent transporter at the apical membrane in renal tubular epithelial cells. Its physiological role is not yet known because studies in octn1 knockout mice are not available. OCTN2 (SLC22A5). OCTN2 was first cloned from human kidney and determined to be the transporter responsible for systemic carnitine deficiency (Tamai et al., 1998). Rat OCTN2 mRNA is expressed predominantly in the cortex, with very little expression in the medulla, and is localized to the apical membrane of the proximal tubule. OCTN2 is a bifunctional transporter. That is, it transports L-carnitine with high affinity in an Na+-dependent manner, whereas, Na+ does not influence OCTN2-mediated transport of organic cations such as TEA. Thus, OCTN2 is thought to function as both an Na+-dependent carnitine transporter and an Na+-independent organic cation transporter. Similar to OCTN1, OCTN2 transport of organic cations is sensitive to pH, suggesting that it may function as an organic cation exchanger. Studies in mice containing a missense mutation in Slc22a5 suggest that organic cations are transported in a secretory direction by OCTN2, whereas carnitine is transported in a reabsorptive direction (Ohashi et al., 2001). Therefore, transport of L-carnitine by OCTN2 is an Na+-dependent electrogenic process. Mutations in OCTN2 have been found to be the cause of primary systemic carnitine deficiency (OMIM 212140) (Nezu et al., 1999). Polymorphisms of OCTs. Polymorphisms of OCTs have been identified in large post-human genome SNP discovery projects (Kerb et al., 2002; Leabman et al., 2003; Shu et al., 2003). OCT1 exhibits the greatest number of amino acid polymorphisms, followed by OCT2 and then OCT3. Furthermore, allele frequencies of OCT1 amino acid variants in human populations generally are greater than those of OCT2 and OCT3 amino acid variants. Functional studies of OCT1 and OCT2 polymorphisms have been performed. OCT1 exhibits five variants with reduced function. These variants may have important implications clinically in terms of hepatic drug disposition and targeting of OCT1 substrates. In particular, individuals with OCT1 variants may have reduced liver uptake of OCT1 substrates and therefore reduced metabolism. Clinical studies need to be performed to ascertain the implications of OCT1 variants to drug disposition and response. For OCT2, several polymorphisms exhibited altered kinetic properties when expressed in Xenopus laevis oocytes. These variants may lead to alterations in renal secretion of OCT2 substrates. Organic Anion Transport. A wide variety of structurally diverse organic anions are secreted in the proximal tubule (Burckhardt and Burckhardt, 2003; Dresser et al., 2001; Wright and Dantzler, 2004). As with organic cation transport, the primary function of organic anion secretion appears to be the removal from the body of xenobiotics, including many weakly acidic drugs [e.g., pravastatin, captopril, p-aminohippurate (PAH), and penicillins] and toxins (e.g., ochratoxin). Organic anion transporters move both hydrophobic and hydrophilic anions but also may interact with cations and neutral compounds. A current model for the transepithelial flux of organic anions in the proximal tubule is shown in Figure 2-12. Two primary transporters on the basolateral membrane mediate the flux of organic anions from interstitial fluid to tubule cell: OAT1 (SLC22A6) and OAT3 (SLC22A8). Energetically, hydrophilic organic anions are transported across the basolateral membrane against an electrochemical gradient in exchange with intracellular a-ketoglutarate, which moves down its concentration gradient from cytosol to blood. The outwardly directed gradient of a-ketoglutarate is maintained at least in part by a basolateral Na+-dicarboxylate transporter (NaDC3). The Na+ gradient that drives NaDC3 is maintained by Na+,K+-ATPase. Transport of small-molecular-weight organic anions by the cloned transporters OAT1 and OAT3 can be driven by a-ketoglutarate. Coupled transport of a-ketoglutarate and small-molecular-weight organic anions (e.g., p-aminohippurate) has been demonstrated in many studies in isolated basolateral membrane vesicles. The molecular pharmacology and molecular biology of OATs have recently been reviewed (Eraly et al., 2004). The mechanism responsible for the apical membrane transport of organic anions from tubule cell cytosol to tubular lumen remains controversial. Some studies suggest that OAT4 may serve as the luminal membrane transporter for organic anions. However, recent studies show that the movement of substrates via this transporter can be driven by exchange with a-ketoglutarate, suggesting that OAT4 may function in the reabsorptive, rather than secretory, flux of organic anions. Other studies have suggested that in the pig kidney, OATV1 serves as an electrogenic facilitated transporter on the apical membrane (Jutabha et al., 2003). The human ortholog of OATV1 is NPT1, or NaPi-1, originally cloned as a phosphate transporter. NPT1 can support the low-affinity transport of hydrophilic organic anions such as PAH. Other transporters that may play a role in transport across the apical membrane include MRP2 and MRP4, multidrug-resistance transporters in the ATP binding cassette family C (ABCC). Both transporters interact with some organic anions and may actively pump their substrates from tubule cell cytosol to tubular lumen. OAT1 (SLC22A6). OAT1 was cloned from rat kidney (Sekine et al., 1997; Sweet et al., 1997). This transporter is greater than 30% identical to OCTs in the SLC22 family. Mouse, human, pig, and rabbit orthologs have been cloned and are approximately 80% identical to human OAT1. Mammalian isoforms of OAT1 vary in length from 545 to 551 amino acids, with features similar to those shown in Figure 2-11. The gene for the human OAT1 is mapped to chromosome 11 and is found in an SLC22 cluster that includes OAT3 and OAT4. There are four splice variants in human tissues, termed OAT1-1, OAT1-2, OAT1-3, and OAT1-4. OAT1-2, which includes a 13-amino-acid deletion, transports PAH at a rate comparable with OAT1-1. These two splice variants use the alternative 5¢-splice sites in exon 9. OAT1-3 and OAT1-4, which result from a 132-bp (44-amino-acid) deletion near the carboxyl terminus of OAT1, do not transport PAH. In humans, rat, and mouse, OAT1 is expressed primarily in the kidney, with some expression in brain and skeletal muscle. Immunohistochemical studies suggest that OAT1 is expressed on the basolateral membrane of the proximal tubule in human and rat, with highest expression in the middle segment, S2. Based on a quantitative polymerase chain reaction (PCR), OAT1 is expressed at a third of the level of OAT3, the other major basolateral membrane organic anion transporter. OAT1 exhibits saturable transport of organic anions such as PAH. This transport is trans-stimulated by other organic anions, including a-ketoglutarate. Thus, the inside negative-potential difference drives the efflux of the dicarboxylate a-ketoglutarate, which, in turn, supports the influx of monocarboxylates such as PAH. Regulation of expression levels of OAT1 in the kidney appears to be controlled by sex steroids. OAT1 generally transports small-molecular-weight organic anions that may be endogenous (e.g., PGE2 and urate) or ingested drugs and toxins. Some neutral compounds are also transported by OAT1 at a lower affinity (e.g., cimetidine). Key residues that contribute to transport by OAT1 include the conserved K394 and R478, which are involved in the PAH-glutarate exchange mechanism. OAT2 (SLC22A7). OAT2 was cloned first from rat liver (and named NLT at the time) (Sekine et al., 1998; Simonson et al., 1994). This transporter has a gender-based tissue distribution between the liver and the kidney in rodents but not in humans, OAT2 is present in both kidney and liver. In the kidney, the transporter is localized to the basolateral membrane of the proximal tubule. Efforts to stimulate organic anion-organic anion exchange via OAT2 have not been successful, leading to the speculation that OAT2 is a basolateral membrane transporter that serves in the reabsorptive flux of organic anions from tubule cell cytosol to interstitial fluids. OAT2 transports many organic anions, including PAH, methotrexate, ochratoxin A, and glutarate. Human, mouse, and rat orthologs of OAT2 have high affinities for the endogenous prostaglandin, PGE2. OAT3 (SLC22A8). OAT3 (SLC22A8) was cloned originally from rat kidney (Kusuhara et al., 1999). Human OAT3 consists of two variants, one of which transports a wide variety of organic anions, including PAH and estrone sulfate. The longer OAT3 in humans, a 568-amino-acid protein, does not support transport. It is likely that the two OAT3 variants are splice variants. Northern blotting suggests that the human ortholog of OAT3 is primarily in the kidney. Mouse and rat orthologs show some expression in the brain and liver. OAT3 mRNA levels are higher than those of OAT1, which in turn are higher than those of OAT2 or OAT4. Human OAT3 is confined to the basolateral membrane of the proximal tubule. OAT3 clearly has overlapping specificities with OAT1, although kinetic parameters differ. For example, estrone sulfate is transported by both OAT1 and OAT3, but OAT3 has a much higher affinity in comparison with OAT1. The weak base cimetidine (an H2-receptor antagonist) is transported with high affinity by OAT1, whereas the cation TEA is not transported. Domains and residues involved in the charge specificity of OAT3 have been identified in several studies. Interestingly, changing two basic amino acid residues in OAT3 (R454D and K370A) shifts the charge specificity of OAT3 from anionic to cationic. Like OAT1, OAT3 appears to be an exchanger that couples the outward flux of a-ketoglutarate to the inward flux of organic anions: The inside negative-potential difference repels a-ketoglutarate from the cells via OAT3, which in turn transports its substrates against a concentration gradient into the tubule cell cytosol. OAT4 (SLC22A9). OAT4 (SLC22A9) was cloned from a human kidney cDNA library (Cha et al., 2000). Quantitative PCR indicates that the expression level of OAT4 in human kidneys is approximately 5% to 10% of the level of OAT1 and OAT3 and is comparable with OAT2. OAT4 is expressed in human kidney and placenta; in human kidney, OAT4 is present on the luminal membrane of the proximal tubule. At first, OAT4 was thought to be involved in the second step of secretion of organic anions, i.e., transport across the apical membrane from cell to tubular lumen. However, recent studies demonstrate that organic anion transport by OAT4 can be stimulated by transgradients of a-ketoglutarate (Ekaratanawong et al., 2004), suggesting that OAT4 may be involved in the reabsorption of organic anions from tubular lumen into cell. The specificity of OAT4 is narrow but includes estrone sulfate and PAH. Interestingly, the affinity for PAH is low (>1 mM). Collectively, emerging studies suggest that OAT4 may be involved not in secretory flux of organic anions but in reabsorption instead. Other Anion Transporters. URAT1 (SLC22A12), first cloned from human kidney, is a kidney-specific transporter confined to the apical membrane of the proximal tubule (Enomoto et al., 2002). Data suggest that URAT1 is primarily responsible for urate reabsorption, mediating electroneutral urate transport that can be trans-stimulated by Cl- gradients. The mouse ortholog of URAT1 is involved in the renal secretory flux of organic anions including benzylpenicillin and urate. NPT1 (SLC17A1), cloned originally as a phosphate transporter in humans, is expressed in abundance on the luminal membrane of the proximal tubule as well as in the brain (Werner et al., 1991). NPT1 transports PAH, probenecid, and penicillin G. It appears to be part of the system involved in organic anion efflux from tubule cell to lumen. MRP2 (ABCC2), an ABC transporter, initially called the GS-X pump (Ishikawa et al., 1990), has been considered to be the primary transporter involved in efflux of many drug conjugates such as glutathione conjugates across the canalicular membrane of the hepatocyte. However, MRP2 is also found on the apical membrane of the proximal tubule, where it is thought to play a role in the efflux of organic anions into the tubular lumen. Its role in the kidney may be to secrete glutathione conjugates of drugs, but it also may support the translocation (with glutathione) of various nonconjugated substrates. In general, MRP2 transports larger, bulkier compounds than do most of the organic anion transporters in the SLC22 family. MRP4 (ABCC4) is found on the apical membrane of the proximal tubule and transports a wide array of conjugated anions, including glucuronide and glutathione conjugates. However, unlike MRP2, MRP4 appears to interact with various drugs, including methotrexate, cyclic nucleotide analogs, and antiviral nucleoside analogs. It is possible that MRP4 is involved in the apical flux of many drugs from cell to tubule lumen. Other MRP efflux transporters also have been identified in human kidney, including MRP3 and MRP6, both on the basolateral membrane. Their roles in the kidney are not yet known. Polymorphisms of OATs. Polymorphisms in OAT1 and OAT3 have been identified in ethnically diverse human populations. Two amino acid polymorphisms (allele frequencies greater than 1%) in OAT1 have been identified in African-American populations (OAT1-R50H). Three amino acid polymorphisms and seven rare amino acid variants in OAT3 have been identified in ethnically diverse U.S. populations (see http://www.pharmgkb.org). TRANSPORTERS INVOLVED IN PHARMACODYNAMICS: DRUG ACTION IN THE BRAIN Neurotransmitters are packaged in vesicles in presynpatic neurons, released in the synapse by fusion of the vesicles with the plasma membrane, and, excepting acetylcholine, are then taken back into the presynaptic neurons or postsynaptic cells (see Chapter 6). Transporters involved in the neuronal reuptake of the neurotransmitters and the regulation of their levels in the synaptic cleft belong to two major superfamilies, SLC1 and SLC6. Transporters in both families play roles in reuptake of g-aminobutyric acid (GABA), glutamate, and the monoamine neurotransmitters norepinephrine, serotonin, and dopamine. These transporters may serve as pharmacologic targets for neuropsychiatric drugs. SLC6 family members localized in the brain and involved in the reuptake of neurotransmitters into presynaptic neurons include the norepinephrine transporters (NET, SLC6A2), the dopamine transporter (DAT, SLC6A3), the serotonin transporter (SERT, SLC6A4), and several GABA reuptake transporters (GAT1, GAT2, and GAT3) (Chen et al., 2004; Hediger, 2004; Elliott and Beveridge, 2005). Each of these transporters appears to have 12 transmembrane secondary structures and a large extracellular loop with glycosylation sites between transmembrane domains 3 and 4. These proteins are typically approximately 600 amino acids in length. SLC6 family members depend on the Na+ gradient to actively transport their substrates into cells. Cl- is also required, although to a variable extent depending on the family member. Residues and domains that form the substrate recognition and permeation pathways are currently being identified. Through reuptake mechanisms, the neurotransmitter transporters in the SLC6A family regulate the concentrations and dwell times of neurotransmitters in the synaptic cleft; the extent of transmitter uptake also influences subsequent vesicular storage of transmitters. It is important to note that many of these transporters are present in other tissues (e.g., kidney and platelets) and may serve other roles. Further, the transporters can function in the reverse direction. That is, the transporters can export neurotransmitters in an Na+-independent fashion. The characteristics of each member of the SLC6A family of transporters that play a role in reuptake of monoamine neurotransmitters and GABA merit a brief description. SLC6A1 (GAT1), SLC6A11 (GAT3), and SLC6A13 (GAT2). GAT1 (599 amino acids) is the most important GABA transporter in the brain, expressed in GABAergic neurons and found largely on presynaptic neurons (Chen et al., 2004). GAT1 is found in abundance in the neocortex, cerebellum, basal ganglia, brainstem, spinal cord, retina, and olfactory bulb. GAT3 is found only in the brain, largely in glial cells. GAT2 is found in peripheral tissues, including the kidney and liver, and within the CNS in the choroid plexus and meninges. GAT1, GAT2, and GAT3 are approximately 50% identical in amino acid sequence. Functional analysis indicates that GAT1 transports GABA with a 2:1 Na+:GABA- stoichiometry. Cl- is required. Residues and domains responsible for the recognition of GABA and subsequent translocation have been identified: Tyr140 appears to be crucial for binding GABA. Physiologically, GAT1 appears to be responsible for regulating the interaction of GABA at receptors. The presence of GAT2 in the choroid plexus and its absence in presynaptic neurons suggest that this transporter may play a primary role in maintaining the homeostasis of GABA in the CSF. GAT1 and GAT3 are drug targets. GAT1 is the target of the antiepileptic drug tiagabine, which presumably acts to increase GABA levels in the synaptic cleft of GABAergic neurons by inhibiting the reuptake of GABA. GAT3 is the target for the nipecotic acid derivatives that are anticonvulsants. SLC6A2 (NET). NET (617 amino acids) is found in central and peripheral nervous tissues as well as in adrenal chromaffin tissue (Chen et al., 2004). In the brain, NET colocalizes with neuronal markers, consistent with a role in reuptake of monoamine neurotransmitters. The transporter functions in the Na+-dependent reuptake of norepinephrine and dopamine and as a higher-capacity norepinephrine channel. A major role of NET is to limit the synaptic dwell time of norepinephrine and to terminate its actions, salvaging norepinephrine for subsequent repackaging. NET knockout mice exhibit a prolonged synaptic half-life of norepinephrine (Xu et al., 2000). Ultimately, through its reuptake function, NET participates in the regulation of many neurological functions, including memory and mood. NET serves as a drug target; the antidepressant desipramine is considered a selective inhibitor of NET. Other drugs that interact with NET include other tricyclic antidepressants and cocaine. Orthostatic intolerance, a rare familial disorder characterized by an abnormal blood pressure and heart rate response to changes in posture, has been associated with a mutation in NET. SLC6A3 (DAT). DAT is located primarily in the brain in dopaminergic neurons. Although present on presynaptic neurons at the neurosynapatic junction, DAT is also present in abundance along the neurons, away from the synaptic cleft. This distribution suggests that DAT may play a role in clearance of excess dopamine in the vicinity of neurons. The primary function of DAT is the reuptake dopamine, terminating its actions, although DAT also weakly interacts with norepinephrine. Physiologically, DAT is involved in the various functions that are attributed to the dopaminergic system, including mood, behavior, reward, and cognition. The half-life of dopamine in the extracellular spaces of the brain is prolonged considerably in DAT knockout mice (Uhl, 2003), which are hyperactive and have sleep disorders. Drugs that interact with DAT include cocaine and its analogs, amphetamines, and the neurotoxin MPTP. SLC6A4 (SERT). SERT is located in peripheral tissues and in the brain along extrasynaptic axonal membranes (Chen et al., 2004; Olivier et al., 2000). SERT clearly plays a role in the reuptake and clearance of serotonin in the brain. Like the other SLC6A family members, SERT transports its substrates in an Na+-dependent fashion and is dependent on Cl- and possibly on the countertransport of K+. Substrates of SERT include serotonin (5-HT), various tryptamine derivatives, and neurotoxins such as 3,4-methylene-dioxymethamphetamine (MDMA; ecstasy) and fenfluramine. The serotonin transporter has been one of the most widely studied proteins in the human genome. First, it is the specific target of the antidepressants in the selective serotonin reuptake inhibitor class (e.g., fluoxetine and paroxetine) and one of several targets of tricyclic antidepressants (e.g., amitriptyline). Further, because of the important role of serotonin in neurological function and behavior, genetic variants of SERT have been associated with an array of behavioral and neurological disorders. In particular, a common promoter region variant that alters the length of the upstream region of SLC6A4 has been the subject of many studies. The short form of the variant results in a reduced rate of transcription of SERT in comparison with the long form. These differences in the rates of transcription alter the quantity of mRNA and, ultimately, the expression and activity of SERT. The short form has been associated with a variety of neuropsychiatric disorders (Lesch et al., 1996). The precise mechanism by which a reduced activity of SERT, caused by either a genetic variant or an antidepressant, ultimately affects behavior, including depression, is not known.

Teknologi Pengolahan Limbah B3

Teknologi Pengolahan Limbah B3 by Wahyu Hidayat on 02/01/08 at 6:43 pm | 117 Comments | | Definisi limbah B3 berdasarkan BAPEDAL (1995) ialah setiap bahan sisa (limbah) suatu kegiatan proses produksi yang mengandung bahan berbahaya dan beracun (B3) karena sifat (toxicity, flammability, reactivity, dan corrosivity) serta konsentrasi atau jumlahnya yang baik secara langsung maupun tidak langsung dapat merusak, mencemarkan lingkungan, atau membahayakan kesehatan manusia. Berdasarkan sumbernya, limbah B3 dapat diklasifikasikan menjadi: o Primary sludge, yaitu limbah yang berasal dari tangki sedimentasi pada pemisahan awal dan banyak mengandung biomassa senyawa organik yang stabil dan mudah menguap o Chemical sludge, yaitu limbah yang dihasilkan dari proses koagulasi dan flokulasi o Excess activated sludge, yaitu limbah yang berasal dari proses pengolahan dengn lumpur aktif sehingga banyak mengandung padatan organik berupa lumpur dari hasil proses tersebut o Digested sludge, yaitu limbah yang berasal dari pengolahan biologi dengan digested aerobic maupun anaerobic di mana padatan/lumpur yang dihasilkan cukup stabil dan banyak mengandung padatan organik. Limbah B3 dikarakterisasikan berdasarkan beberapa parameter yaitu total solids residue (TSR), kandungan fixed residue (FR), kandungan volatile solids (VR), kadar air (sludge moisture content), volume padatan, serta karakter atau sifat B3 (toksisitas, sifat korosif, sifat mudah terbakar, sifat mudah meledak, beracun, serta sifat kimia dan kandungan senyawa kimia). Contoh limbah B3 ialah logam berat seperti Al, Cr, Cd, Cu, Fe, Pb, Mn, Hg, dan Zn serta zat kimia seperti pestisida, sianida, sulfida, fenol dan sebagainya. Cd dihasilkan dari lumpur dan limbah industri kimia tertentu sedangkan Hg dihasilkan dari industri klor-alkali, industri cat, kegiatan pertambangan, industri kertas, serta pembakaran bahan bakar fosil. Pb dihasilkan dari peleburan timah hitam dan accu. Logam-logam berat pada umumnya bersifat racun sekalipun dalam konsentrasi rendah. Daftar lengkap limbah B3 dapat dilihat di PP No. 85 Tahun 1999: Pengelolaan Limbah Bahan Berbahaya dan Beracun (B3). Silakan klik link tersebut untuk daftar lengkap yang juga mencakup peraturan resmi dari Pemerintah Indonesia. Penanganan atau pengolahan limbah padat atau lumpur B3 pada dasarnya dapat dilaksanakan di dalam unit kegiatan industri (on-site treatment) maupun oleh pihak ketiga (off-site treatment) di pusat pengolahan limbah industri. Apabila pengolahan dilaksanakan secara on-site treatment, perlu dipertimbangkan hal-hal berikut: o jenis dan karakteristik limbah padat yang harus diketahui secara pasti agar teknologi pengolahan dapat ditentukan dengan tepat; selain itu, antisipasi terhadap jenis limbah di masa mendatang juga perlu dipertimbangkan o jumlah limbah yang dihasilkan harus cukup memadai sehingga dapat menjustifikasi biaya yang akan dikeluarkan dan perlu dipertimbangkan pula berapa jumlah limbah dalam waktu mendatang (1 hingga 2 tahun ke depan) o pengolahan on-site memerlukan tenaga tetap (in-house staff) yang menangani proses pengolahan sehingga perlu dipertimbangkan manajemen sumber daya manusianya o peraturan yang berlaku dan antisipasi peraturan yang akan dikeluarkan Pemerintah di masa mendatang agar teknologi yang dipilih tetap dapat memenuhi standar Teknologi Pengolahan Terdapat banyak metode pengolahan limbah B3 di industri, tiga metode yang paling populer di antaranya ialah chemical conditioning, solidification/Stabilization, dan incineration. 1. Chemical Conditioning Salah satu teknologi pengolahan limbah B3 ialah chemical conditioning. TUjuan utama darichemical conditioning ialah: 1. menstabilkan senyawa-senyawa organik yang terkandung di dalam lumpur 2. mereduksi volume dengan mengurangi kandungan air dalam lumpur 3. mendestruksi organisme patogen 4. memanfaatkan hasil samping proses chemical conditioning yang masih memiliki nilai ekonomi seperti gas methane yang dihasilkan pada proses digestion 5. mengkondisikan agar lumpur yang dilepas ke lingkungan dalam keadaan aman dan dapat diterima lingkungan Chemical conditioning terdiri dari beberapa tahapan sebagai berikut: 6. Concentration thickening Tahapan ini bertujuan untuk mengurangi volume lumpur yang akan diolah dengan cara meningkatkan kandungan padatan. Alat yang umumnya digunakan pada tahapan ini ialahgravity thickener dan solid bowl centrifuge. Tahapan ini pada dasarnya merupakan tahapan awal sebelum limbah dikurangi kadar airnya pada tahapan de-watering selanjutnya. Walaupun tidak sepopuler gravity thickener dan centrifuge, beberapa unit pengolahan limbah menggunakan proses flotation pada tahapan awal ini. 7. Treatment, stabilization, and conditioning Tahapan kedua ini bertujuan untuk menstabilkan senyawa organik dan menghancurkan patogen. Proses stabilisasi dapat dilakukan melalui proses pengkondisian secara kimia, fisika, dan biologi. Pengkondisian secara kimia berlangsung dengan adanya proses pembentukan ikatan bahan-bahan kimia dengan partikel koloid. Pengkondisian secara fisika berlangsung dengan jalan memisahkan bahan-bahan kimia dan koloid dengan cara pencucian dan destruksi. Pengkondisian secara biologi berlangsung dengan adanya proses destruksi dengan bantuan enzim dan reaksi oksidasi. Proses-proses yang terlibat pada tahapan ini ialahlagooning, anaerobic digestion, aerobic digestion, heat treatment, polyelectrolite flocculation,chemical conditioning, dan elutriation. 8. De-watering and drying De-watering and drying bertujuan untuk menghilangkan atau mengurangi kandungan air dan sekaligus mengurangi volume lumpur. Proses yang terlibat pada tahapan ini umumnya ialah pengeringan dan filtrasi. Alat yang biasa digunakan adalah drying bed, filter press,centrifuge, vacuum filter, dan belt press. 9. Disposal Disposal ialah proses pembuangan akhir limbah B3. Beberapa proses yang terjadi sebelum limbah B3 dibuang ialah pyrolysis, wet air oxidation, dan composting. Tempat pembuangan akhir limbah B3 umumnya ialah sanitary landfill, crop land, atau injection well. Solidification/Stabilization Di samping chemical conditiong, teknologi solidification/stabilization juga dapat diterapkan untuk mengolah limbah B3. Secara umum stabilisasi dapat didefinisikan sebagai proses pencapuran limbah dengan bahan tambahan (aditif) dengan tujuan menurunkan laju migrasi bahan pencemar dari limbah serta untuk mengurangi toksisitas limbah tersebut. Sedangkan solidifikasi didefinisikan sebagai proses pemadatan suatu bahan berbahaya dengan penambahan aditif. Kedua proses tersebut seringkali terkait sehingga sering dianggap mempunyai arti yang sama. Proses solidifikasi/stabilisasi berdasarkan mekanismenya dapat dibagi menjadi 6 golongan, yaitu: 0. Macroencapsulation, yaitu proses dimana bahan berbahaya dalam limbah dibungkus dalam matriks struktur yang besar 1. Microencapsulation, yaitu proses yang mirip macroencapsulation tetapi bahan pencemar terbungkus secara fisik dalam struktur kristal pada tingkat mikroskopik 2. Precipitation 3. Adsorpsi, yaitu proses dimana bahan pencemar diikat secara elektrokimia pada bahan pemadat melalui mekanisme adsorpsi. 4. Absorbsi, yaitu proses solidifikasi bahan pencemar dengan menyerapkannya ke bahan padat 5. Detoxification, yaitu proses mengubah suatu senyawa beracun menjadi senyawa lain yang tingkat toksisitasnya lebih rendah atau bahkan hilang sama sekali Teknologi solidikasi/stabilisasi umumnya menggunakan semen, kapur (CaOH2), dan bahan termoplastik. Metoda yang diterapkan di lapangan ialah metoda in-drum mixing, in-situ mixing, danplant mixing. Peraturan mengenai solidifikasi/stabilitasi diatur oleh BAPEDAL berdasarkan Kep-03/BAPEDAL/09/1995 dan Kep-04/BAPEDAL/09/1995. Incineration Teknologi pembakaran (incineration ) adalah alternatif yang menarik dalam teknologi pengolahan limbah. Insinerasi mengurangi volume dan massa limbah hingga sekitar 90% (volume) dan 75% (berat). Teknologi ini sebenarnya bukan solusi final dari sistem pengolahan limbah padat karena pada dasarnya hanya memindahkan limbah dari bentuk padat yang kasat mata ke bentuk gas yang tidak kasat mata. Proses insinerasi menghasilkan energi dalam bentuk panas. Namun, insinerasi memiliki beberapa kelebihan di mana sebagian besar dari komponen limbah B3 dapat dihancurkan dan limbah berkurang dengan cepat. Selain itu, insinerasi memerlukan lahan yang relatif kecil. Aspek penting dalam sistem insinerasi adalah nilai kandungan energi (heating value) limbah. Selain menentukan kemampuan dalam mempertahankan berlangsungnya proses pembakaran, heating value juga menentukan banyaknya energi yang dapat diperoleh dari sistem insinerasi. Jenis insinerator yang paling umum diterapkan untuk membakar limbah padat B3 ialah rotary kiln, multiple hearth, fluidized bed, open pit, single chamber, multiple chamber, aqueous waste injection, dan starved air unit. Dari semua jenis insinerator tersebut, rotary kiln mempunyai kelebihan karena alat tersebut dapat mengolah limbah padat, cair, dan gas secara simultan. Penanganan Limbah B3 Hazardous Material Container Limbah B3 harus ditangani dengan perlakuan khusus mengingat bahaya dan resiko yang mungkin ditimbulkan apabila limbah ini menyebar ke lingkungan. Hal tersebut termasuk proses pengemasan, penyimpanan, dan pengangkutannya. Pengemasan limbah B3 dilakukan sesuai dengan karakteristik limbah yang bersangkutan. Namun secara umum dapat dikatakan bahwa kemasan limbah B3 harus memiliki kondisi yang baik, bebas dari karat dan kebocoran, serta harus dibuat dari bahan yang tidak bereaksi dengan limbah yang disimpan di dalamnya. Untuk limbah yang mudah meledak, kemasan harus dibuat rangkap di mana kemasan bagian dalam harus dapat menahan agar zat tidak bergerak dan mampu menahan kenaikan tekanan dari dalam atau dari luar kemasan. Limbah yang bersifat self-reactive dan peroksida organik juga memiliki persyaratan khusus dalam pengemasannya. Pembantalan kemasan limbah jenis tersebut harus dibuat dari bahan yang tidak mudah terbakar dan tidak mengalami penguraian (dekomposisi) saat berhubungan dengan limbah. Jumlah yang dikemas pun terbatas sebesar maksimum 50 kg per kemasan sedangkan limbah yang memiliki aktivitas rendah biasanya dapat dikemas hingga 400 kg per kemasan. Limbah B3 yang diproduksi dari sebuah unit produksi dalam sebuah pabrik harus disimpan dengan perlakuan khusus sebelum akhirnya diolah di unit pengolahan limbah. Penyimpanan harus dilakukan dengan sistem blok dan tiap blok terdiri atas 2×2 kemasan. Limbah-limbah harus diletakkan dan harus dihindari adanya kontak antara limbah yang tidak kompatibel. Bangunan penyimpan limbah harus dibuat dengan lantai kedap air, tidak bergelombang, dan melandai ke arah bak penampung dengan kemiringan maksimal 1%. Bangunan juga harus memiliki ventilasi yang baik, terlindung dari masuknya air hujan, dibuat tanpa plafon, dan dilengkapi dengan sistem penangkal petir. Limbah yang bersifat reaktif atau korosif memerlukan bangunan penyimpan yang memiliki konstruksi dinding yang mudah dilepas untuk memudahkan keadaan darurat dan dibuat dari bahan konstruksi yang tahan api dan korosi. Mengenai pengangkutan limbah B3, Pemerintah Indonesia belum memiliki peraturan pengangkutan limbah B3 hingga tahun 2002. Namun, kita dapat merujuk peraturan pengangkutan yang diterapkan di Amerika Serikat. Peraturan tersebut terkait dengan hal pemberian label, analisa karakter limbah, pengemasan khusus, dan sebagainya. Persyaratan yang harus dipenuhi kemasan di antaranya ialah apabila terjadi kecelakaan dalam kondisi pengangkutan yang normal, tidak terjadi kebocoran limbah ke lingkungan dalam jumlah yang berarti. Selain itu, kemasan harus memiliki kualitas yang cukup agar efektivitas kemasan tidak berkurang selama pengangkutan. Limbah gas yang mudah terbagak harus dilengkapi dengan head shields pada kemasannya sebagai pelindung dan tambahan pelindung panas untuk mencegah kenaikan suhu yang cepat. Di Amerika juga diperlakukan rute pengangkutan khusus selain juga adanya kewajiban kelengkapan Material Safety Data Sheets (MSDS) yang ada di setiap truk dan di dinas pemadam kebarakan. Secured Landfill. Faktor hidrogeologi, geologi lingkungan, topografi, dan faktor-faktor lainnya harus diperhatikan agar secured landfill tidak merusak lingkungan. Pemantauan pasca-operasi harus terus dilakukan untuk menjamin bahwa badan air tidak terkontaminasi oleh limbah B3. Pembuangan Limbah B3 (Disposal) Sebagian dari limbah B3 yang telah diolah atau tidak dapat diolah dengan teknologi yang tersedia harus berakhir pada pembuangan (disposal). Tempat pembuangan akhir yang banyak digunakan untuk limbah B3 ialah landfill (lahan urug) dan disposal well (sumur pembuangan). Di Indonesia, peraturan secara rinci mengenai pembangunan lahan urug telah diatur oleh Badan Pengendalian Dampak Lingkungan (BAPEDAL) melalui Kep-04/BAPEDAL/09/1995. Landfill untuk penimbunan limbah B3 diklasifikasikan menjadi tiga jenis yaitu: (1) secured landfill double liner, (2) secured landfill single liner, dan (3) landfill clay liner dan masing-masing memiliki ketentuan khusus sesuai dengan limbah B3 yang ditimbun. Dimulai dari bawah, bagian dasar secured landfill terdiri atas tanah setempat, lapisan dasar, sistem deteksi kebocoran, lapisan tanah penghalang, sistem pengumpulan dan pemindahan lindi (leachate), dan lapisan pelindung. Untuk kasus tertentu, di atas dan/atau di bawah sistem pengumpulan dan pemindahan lindi harus dilapisi geomembran. Sedangkan bagian penutup terdiri dari tanah penutup, tanah tudung penghalang, tudung geomembran, pelapis tudung drainase, dan pelapis tanah untuk tumbuhan dan vegetasi penutup. Secured landfill harus dilapisi sistem pemantauan kualitas air tanah dan air pemukiman di sekitar lokasi agar mengetahui apakah secured landfill bocor atau tidak. Selain itu, lokasi secured landfill tidak boleh dimanfaatkan agar tidak beresiko bagi manusia dan habitat di sekitarnya. Deep Injection Well. Pembuangan limbah B3 melalui metode ini masih mejadi kontroversi dan masih diperlukan pengkajian yang komprehensif terhadap efek yang mungkin ditimbulkan. Data menunjukkan bahwa pembuatan sumur injeksi di Amerika Serikat paling banyak dilakukan pada tahun 1965-1974 dan hampir tidak ada sumur baru yang dibangun setelah tahun 1980. Sumur injeksi atau sumur dalam (deep well injection) digunakan di Amerika Serikat sebagai salah satu tempat pembuangan limbah B3 cair (liquid hazardous wastes). Pembuangan limbah ke sumur dalam merupakan suatu usaha membuang limbah B3 ke dalam formasi geologi yang berada jauh di bawah permukaan bumi yang memiliki kemampuan mengikat limbah, sama halnya formasi tersebut memiliki kemampuan menyimpan cadangan minyak dan gas bumi. Hal yang penting untuk diperhatikan dalam pemilihan tempat ialah strktur dan kestabilan geologi serta hidrogeologi wilayah setempat. Limbah B3 diinjeksikan se dalam suatu formasi berpori yang berada jauh di bawah lapisan yang mengandung air tanah. Di antara lapisan tersebut harus terdapat lapisan impermeable seperti shaleatau tanah liat yang cukup tebal sehingga cairan limbah tidak dapat bermigrasi. Kedalaman sumur ini sekitar 0,5 hingga 2 mil dari permukaan tanah. Tidak semua jenis limbah B3 dapat dibuang dalam sumur injeksi karena beberapa jenis limbah dapat mengakibatkan gangguan dan kerusakan pada sumur dan formasi penerima limbah. Hal tersebut dapat dihindari dengan tidak memasukkan limbah yang dapat mengalami presipitasi, memiliki partikel padatan, dapat membentuk emulsi, bersifat asam kuat atau basa kuat, bersifat aktif secara kimia, dan memiliki densitas dan viskositas yang lebih rendah daripada cairan alami dalam formasi geologi. Hingga saat ini di Indonesia belum ada ketentuan mengenai pembuangan limbah B3 ke sumur dalam (deep injection well). Ketentuan yang ada mengenai hal ini ditetapkan oleh Amerika Serikat dan dalam ketentuan itu disebutkah bahwa: 1. Dalam kurun waktu 10.000 tahun, limbah B3 tidak boleh bermigrasi secara vertikal keluar dari zona injeksi atau secara lateral ke titik temu dengan sumber air tanah. 2. Sebelum limbah yang diinjeksikan bermigrasi dalam arah seperti disebutkan di atas, limbah telah mengalami perubahan higga tidak lagi bersifat berbahaya dan beracun. Referensi: Pengelolaan Limbah Industri – Prof. Tjandra Setiadi, Wikipedia, US EPA