Daniel W. Nebert, MD

 

Daniel (“Dan”) Walter Nebert is an American physician-scientist, molecular biologist, and geneticist. He has authored/coauthored publications in fields of biochemistry, molecular biology, pediatrics, developmental biology, pharmacology, drug metabolism, toxicology, mouse genetics, human genetics, evolutionary genomics, gene nomenclature, and cancer.

Nebert attended Wesleyan University, Connecticut, and University of Oregon Medical School, Portland, receiving BA, MS and MD degrees (1956–64). After pediatric internship and residency at UCLA Health Sciences Center, Los Angeles (1964–66), Nebert was postdoctoral fellow in the National Cancer Institute (NCI), National Institutes of Health (NIH), Bethesda, MD (1966–68), following which he carried out and directed basic and clinical research in the National Institute of Child Health & Human Development (NICHD) for 21 years.

Nebert became Professor, Department of Environmental Health at University of Cincinnati College of Medicine (1989), and Adjunct Professor in the Human Genetics Division, Department of Pediatrics and Molecular & Developmental Biology at Cincinnati Children’s Hospital Medical Center (1991). He retired as Emeritus in 2013. Nebert helped reshape the Department of Environmental Health from 20th-century basic toxicology to 21st-century environmental molecular genetics and genomics.

Early life and education

Dan Nebert was born in September 1938, in Portland, Oregon — the second of four children; his parents were Walter Francis Nebert (electrician foreman) and Marie Sophie Schick. All four grandparents were descendants of Austria-Bohemia (from Sudetenland and Vienna). “Danny” grew up in the small farming community of Garden Home, OR. Nebert’s earliest childhood memories included: perceiving “war in Europe and the Pacific”; being warned of possible balloon-firebombs, enemy strafing aircraft, and bombs attached to fishing glass-floats in the Pacific Ocean surf; growing his own “victory garden” (ages 4–6); and living in awe of the stars, planets and vastness of the universe. He was convinced that he had come from another planet and was not related to his other family members (his hair was white-blonde while everyone else had dark hair).

Nebert attended Garden Home Grade School (1944–52). For the last 2​¹⁄₂ years he founded, and was editor of, a weekly ‘student news’ newspaper, which included a comic strip. He was valedictorian of his 8th-grade class. Encouraged by his mother, he received eight years of classical piano lessons, following which he began writing music. At age 7, Nebert’s music teacher determined he had “near perfect pitch.” Two of his songs were recorded in 1956 at Castle Recording Studios in Hollywood.

At Beaverton High School, Nebert finished among the top twelve in a class of 260. His American History teacher, Ted Van Buren, strongly urged him to consider a career in government and history. This included a week-long summer course, “Beaver Boys State,” in Corvallis, OR — where he studied geopolitics and government. Hobbies included art (pencil drawing and oil painting; he won First Prize in the Meier & Frank Art Contest, 1951, for a crayon drawing), gardening, landscaping, cooking (won blue ribbon in Salad Division, Tillamook County Fair, 1999), and listening to music — especially Medieval, Renaissance, and Baroque. Before age 18 he wrote fictional short stories which were never accepted for publication in magazines.

Nebert was awarded a General Motors Full Scholarship to attend Wesleyan University (Middletown, CT); he first considered an “academic theology” major, but then concluded it was “not sufficiently quantitative”. Nebert’s organic chemistry teacher Robert Stern (on sabbatical from Yale) and Nebert’s biology mentor Ernst Caspari both urged him to consider medicine as a career — specifically genetics research — “because that’s where an exciting future lies.” Accordingly, Nebert applied to medical school. In a 5-year program at the University of Oregon Medical School (Portland), now named Oregon Health & Science University (OHSU), Nebert obtained a MS degree in biophysics (with Howard S Mason, mentor) plus the MD degree.

Nebert enjoyed competitive sports: in grade school — football, basketball and softball; in high school — basketball, tennis and golf. He was Beaverton High School Medalist in the 1955 Oregon State High School Golf Tournament. In college — basketball, track (high jump; long-distance running) and golf. In the 1964 U.S. Squash Racquets Association International Tournament, Nebert made it to the Class D Finals; he was also on the Bender Jewish Community Center of Greater Washington (Rockville, MD) squash team, one of 12 teams in the Federal League throughout the Greater Washington DC Area (1971–88). He was also a member of the NIH golf team, among 16 teams in the Federal League (1968–88). In NASTAR Olympics downhill slalom ski racing, Keystone, CO (1982), Nebert won a Bronze Medal.

In Los Angeles (1964–66), Nebert served as pediatric intern and resident at UCLA Health Sciences Center (Robert A Ulstrom, Chair of Pediatrics) and Harbor General Hospital (Joseph St. Geme, Chair). Because Nebert was author/coauthor of seven publications by 1966 — including his MS thesis “An electron spin resonance study of normal and neoplastic biological material” — this helped him secure a postdoctoral fellowship at the NCI as Research Fellow in the U.S. Public Health Service, instead of military duty in Southeast Asia.

At the NIH, Harry V Gelboin was mentor for Nebert’s NCI postdoctoral fellowship (1966–68). Nebert was one of the founding members that started a free medical clinic in Washington, D.C. (Southeast Neighborhood Action Board); between 1968 and 1975, up to 85 physicians from the NIH volunteered to work evenings, six days a week, to care for indigent patients (the project folded in 1975, when medical insurance prices went sky-high). Moving to the NICHD, Nebert became Independent Investigator (1968–71), Section Head (1971-75) and Chief of Laboratory of Developmental Pharmacology (1975–89). He organized and hosted three international symposia at The Airlie House, VA (April 1985, 1987 and 1989) on the topics of “cytochrome P450”, and “drug-metabolizing enzymes”, and “gene-environment interactions”.

Nebert then moved to the University Cincinnati Medical Center in December 1989, where he remained until becoming Emeritus in 2013. In 1992 Nebert founded the Center for Environmental Genetics (CEG), the first National Institute of Environmental Health Sciences (NIEHS) Center of Excellence of its kind — focusing on “gene-environment interactions.” As Principal Investigator of the Gene-Environment Interactions Training Program (GEITP; 2008–14), Nebert began a GEITP emailing list of more than 200 colleagues worldwide — which continues to grow, even today. Cutting-edge articles are selected and evaluated by Nebert; many of these commentaries and chats are posted at GeneWhisperer.com.

Nebert has published more than 650 papers in numerous scientific fields. He was recognized by Eugene Garfield [Institute for Scientific Information (ISI)] as “among The 1,000 Contemporary Scientists Most-Cited, 1965-1978,” and “among the Top 0.1% Contemporary Scientists Most-Cited, 1981-1999” — from a compilation of more than 1 million authors in all scientific fields. In 2016 Nebert was ranked by Google Scholar among the top 640 “Most-Cited Scientists/Authors worldwide since 1900” — which includes all fields (Mathematics, Physics, Chemistry, Physiology and Medicine, Literature, Political Science, and Economics). As of January 2020, his Google Scholar h-index was 122 with more than 65,000 citations.

Career and research

Discovery of the AHR transcription factor

As a postdoctoral fellow, Nebert characterized an aryl hydrocarbon hydroxylase (AHH) enzyme assay and AHH induction by many polycyclic aromatic hydrocarbons (PAHs) in fetal hamster cell cultures.^[1][2] Using mouse genetic differences in inducible AHH activity, he then proved^[3] that the AHH enzyme represents a cytochrome P-450, which he named “P₁-450,” now officially “CYP1A1”; the mammalian CYP1 family was later found to comprise three PAH-inducible genes — CYP1A1, CYP1A2 and CYP1B1.^[4] Then, in 1974, using mouse genetic differences in AHH inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; “dioxin”), Nebert with Alan Poland demonstrated a dose-response consistent with receptor regulation; they named it “aryl hydrocarbon receptor” (AHR); with regard to PAH- and dioxin-binding, sensitive mice had a high-affinity AHR, resistant mice a poor-affinity AHR.^[5] Several months later^[6] Nebert published the famous dose-response curve [Fig. 1]

In the Poland lab, following treatment with radiolabeled dioxin, hepatic cytosol accumulation of radiolabel was found to be greatest in C57BL/6 mice, intermediate in B6D2F₁ mice, and least in DBA/2 mice^[7] — a pattern mirroring the strain sensitivity to AHH induction by TCDD, demonstrated earlier^[6] The Chris Bradfield lab cloned the mouse AHR gene^[8] and human AHR gene^[9] and was the first to suggest that aryl hydrocarbon receptor nuclear translocator (ARNT) is a heterodimeric cotranscriptional partner with AHR during gene activation^[8]

Years later it was realized that AHR was the second earliest discovered member of the “basic-helix-loop-helix/per–Arnt–sim” (bHLH/PAS) family,^[10][11][12] which comprises dozens of transcription regulators found throughout all kingdoms of life. The human and mouse genomes each contain about 30 (evolutionarily highly conserved) bHLH/PAS protein-coding genes; this gene subfamily is a subset within the 110 members of the human BHLH gene superfamily. All BHLH proteins “sense” innumerable extracellular and intracellular “signals” — including foreign chemicals, endogenous compounds, gas molecules, redox potential, photons (light), gravity, temperature, and osmotic pressure. After receiving the signal, these BHLH transcription factors mediate downstream-targeting events that are involved in many gene-network pathways and signaling cascades fundamental to life and embryogenesis.

The earliest discovered bHLH/PAS transcription factor was the Drosophila “periodic” (per) locus;^[13] the PER protein affects circadian rhythmicity. In the mid-1980s, characterization of the Drosophila “single-minded” (sim) gene was reported;^[14] the SIM protein responds to developmental signals, giving rise to midline cells of embryonic central nervous system. Later, the mouse Arnt gene was cloned^[15] — following which the term “PAS,” abbreviation for “per-Arnt-sim,” was coined^[16]

In the early 1980s, Nebert found that mouse genetic differences in high- versus poor-affinity AHR are associated with ethanol-caused peritonitis.^[17][18][19] An independent study in chick fetal liver^[20] had noted that nonsteroidal anti-inflammatory benoxaprofen decreased toxicity of planar 3,4,3′,4′-tetrachlorobiphenyl (a known AHR ligand); to Nebert, this finding, combined with the ethanol-caused peritonitis, suggested AHR-mediated involvement in the arachidonic acid cascade. The AHR-mediated response to ethanol-induced peritonitis was therefore postulated to be involved in lipid-mediator (LM) second-messenger pathways (which includes prostaglandins, leukotrienes, eicosanoids, resolvins, neuroprotectins and lipoxins). The strongest evidence in favor of this hypothesis was shown when Nebert with Charles N Serhan — using multiple-reaction monitoring and liquid chromatography-UV-coupled with tandem mass spectrometry-based LM metabololipidomics — demonstrated substantial changes in concentration of leukotriene B₄ and seven other LM metabolites associated with AHR/CYP1-mediated pro-inflammatory and inflammation-resolution pathways^[21]

Using mouse genetic differences in high- versus poor-affinity AHR, the Nebert lab in dozens of studies between 1973 and 2000 characterized the “mouse [Ah] gene battery” and its direct effects on: AHR/CYP1-mediated PAH-, polychlorinated biphenyl- and dioxin-induced carcinogenesis, mutagenesis, toxicity, teratogenesis, mitochondrial H₂O₂ production, and reactive-oxygen species (ROS) formation.^[22][23] The Nebert lab was first to clone and sequence the mouse Cyp1a1 and Cyp1a2 genes^[24] and human CYP1A1 and CYP1A2 genes.^[25][26]

Nebert with Frank J Gonzalez generated the first Ahr(–/–) global knockout mouse.^[27] Nebert’s lab with Steve Potter next created the Cyp1a2(–/–) global knockout.^[28] Then came Cyp1a1(–/–) global^[29] and Cyp1(–/–) conditional^[30][31] knockouts, all three possible Cyp1(–/–) double-knockouts^[32][33] and a viable but abnormal Cyp1(–/–) triple-knockout.^[34] Shared worldwide, these various knockout mouse lines continue to help us understand AHR/CYP1-mediated inflammatory pathways^[12][35] and an appreciation that oral benzo(a)pyrene (BaP)-caused immunotoxicity — as well as the type and location of cancer — is highly dependent on BaP route-of-administration, dose, time, Cyp1 genotype, and target organ.^[36]

The first hint of “AHR/CYP1 signaling during embryogenesis” came from sister-chromatid-exchange (SCE) studies. Nebert with Roger Pedersen showed mouse embryo gestational day (GD)7.5 explant cultures in medium containing BaP and 5-bromodeoxyuridine caused SCEs associated with AHR-responsive, but not AHR-nonresponsive, mouse lines.^[37] Subsequently, Nebert with Anup Dey (NICHD) discovered — in untreated pregnant mice that the zygote 12 hours after (but not before) fertilization — exhibited dramatically elevated levels of AHR-mediated Cyp1a1 mRNA.^[38] This finding has now led to more than 20 years of studies characterizing AHR/CYP1-mediated functions and signaling pathways in embryonic stem (ES) cell cultures and embryoid bodies.^[12]

Other unexpected but intriguing observations included CYP1A1-mediated electrophilic metabolites of PAHs, during exposure of Hepa-1 cells to PAHs, decreased epidermal growth factor (EGF)-binding to EGF cell-surface receptors, whereas occupancy of AHR per se does not affect EGF binding.^[39] Moreover, constitutive Cyp1a1 mRNA is dramatically increased after partial hepatectomy in adult mice, and in 7-day-old mouse embryos, and following retinoic acid-induced differentiation of F9 embryonal carcinoma cells — in each instance in the absence of any foreign chemical inducer.^[40]

Today we know that AHR/CYP1-signaling is involved in an amazing array of genetic networks and subcellular processes critical to life^[12] — including: fundamental early-embryogenesis processes (e.g. cell division, adhesion, migration, cell cycle regulation, germ cell apoptosis, the MID1-PP2A-CDC25B-CDK1 signaling pathway regulating mitosis; ectoderm-to-epithelium transition, transmesoderm-to-osteoblast transition, cavity formation during the morula-to-blastula transition, cardiomyocyte genesis, activator of Rho/Tac GTPases, WNT-signaling pathways, and homeobox-signaling pathways); angiogenesis; organogenesis (e.g. formation of brain and central nervous system, formation of the gastrointestinal tract, pancreas, liver, heart, respiratory tract, kidney, formation and development of immune system, male and female sex organs, cochlea of inner ear, and the eye’s ciliary body); development of the blood cell-forming system (hematopoiesis, activation, as well as suppression of erythroid development); bone formation and osteoclastogenesis; neurogenesis and development of specific neuronal cell-types; the “brain-gut-microbiome” network; participation in the immune response, innate immunity, pro-inflammatory and post-inflammation responses, and immunomodulatory effects; cardiovascular physiology; atherogenesis (plaque formation); hypertension; pancreatic beta-cell regulation, glucose and lipid metabolism, hyperlipidemia, and hepatic steatosis; serum testosterone levels, spermatogenesis, fertility and degenerative changes in testis; mammary gland duct cell epithelial hyperplasia; endometriosis; disruption of GABA-ergic transmission defects; skin barrier physiology; atopic dermatitis; circadian rhythmicity; DNA synthesis and DNA repair; metabolic activation and detoxication of many foreign chemicals; DNA-adduct formation involving mutagenic or toxic metabolites; mutagenesis; mitochondrial reactive-oxygen-species (ROS) formation, as well as anti-oxidant protection against ROS formation; mitochondrial H₂O₂ production; crosstalk with hypoxia and hypoxia-inducible factor (HIF)-signaling pathways; transforming growth factor-signaling pathways, as well as growth suppression; tumor initiation; tumor promotion; transgenerational inheritance and epigenetic effects; chromatin remodeling; histone modification; and aging-related and degenerative diseases.

Pharmacogenomics and genetic prediction of drug response

Following Motulsky’s prediction^[41] and Vogel naming the field “pharmacogenetics”^[42] — it became appreciated that each individual patient’s response to any drug will be largely dependent on that subject’s genetic make-up, i.e. each person’s genetic architecture. After the Human Genome Project was initiated in 1990, the term “pharmacogenetics” (‘gene-drug interactions’) transformed into “pharmacogenomics” (‘genome-drug interactions’).

From the 1960s onward, most reviews on the topic of “genetic contribution to drug response” were felt by Nebert as being too one-dimensional; in other words, an individual’s response to virtually every drug would most likely be far more complex than being caused by just one or a few single-nucleotide variants (SNVs) in an entire haploid genome — considering the holistic nature of each unique human being. From Nebert’s earliest invited review on the topic^[43] to later reviews^[44][45][46] to his latest with Ge Zhang^[47] the complexity to predict individual drug response has evolved, along with our growing knowledge of complexity of the human genome. As concluded in Nebert’s recent book chapter with Ge Zhang^[48] each patient’s response to a drug is now considered to be the combination of [a] genetics, [b] epigenetic effects, [c] endogenous influences, [d] environmental exposures, and [e] each person’s gut microbiome. With the exception of “genetics” (i.e. each person’s germline DNA sequence), the latter four categories are not constant, but rather should be viewed as continuously changing throughout one’s lifetime.^[48]

Spearheading standardized gene nomenclature

With purification of many P450 proteins and creation of antibodies (1970s), each laboratory (working with rat, rabbit or mouse) independently gave each enzyme its own pet name. By the mid-1980s, Nebert felt that this haphazard approach would soon lead to chaos — and would be confusing especially to graduate students and postdoctoral fellows just entering the field, as well as established investigators who wished to learn more about “cytochrome P450 research.”

Also in the early- to mid-1980s, clones of P450 cDNAs began to be sequenced, from which translated amino-acid sequences could be deduced. Intriguingly, if one aligned the protein sequences of P450 proteins from Pseudomonas, yeast, and eight vertebrates including human — the highly conserved cysteinyl-containing peptide involved in the heme-binding enzyme active-site became evident.^[49] This high degree of similarity in the P450 protein consensus sequence between bacteria and human fascinated Nebert.

Given Nebert’s keen interest in evolution from early childhood, and believing there would soon be an explosion in number of gene sequences of many genomes, he suggested that genes in families and subfamilies might be named by a standardized nomenclature system — based on evolutionary divergence. Moreover, the “root,” or “symbol,” for each gene in each superfamily should be the same as that of the original ancestral gene (if that could be determined). The other challenge was to bring “the Principal Investigators of all major P450 purification labs” together and invite them to be coauthors on a standardized gene nomenclature publication.

“P450” was the first “root” chosen for the gene superfamily, and amino-acid sequences of known P450 proteins were compared for “percent similarity” — as described in the first nomenclature paper with 13 coauthors.^[50] Subsequently, it was decided the “gene root name” should be only letters, not a combination of letters and numbers; Nebert suggested “CYP” as the root, and this was agreed upon by all coauthors of the second nomenclature update.^[51]

Gene families and subfamilies needed to be categorized within the superfamily. The original “cut-off” for members within one family was >40% identity, or similarity; P450 protein sequences having <40% similarity would represent CYP genes of different families. The original cut-off for members within one subfamily was >60% similarity. Of course, many complications arose, and each new gene had to be manually curated and decisions made. The earliest method of visualizing DNA or protein sequence similarities/differences was the unweighted pair-group method with arithmetic mean (UPGMA), a simple agglomerative (bottom-up) hierarchical clustering method [Fig. 2]. Today, many new algorithms have been developed — in this expanding field of evolutionary divergence analysis.

In 1996 was the last publication^[4] before the total number of CYP genes became unwieldy for journal updates. David R Nelson at University Tennessee Health Sciences Center in Memphis volunteered to become curator of the CYP gene nomenclature homepage website and continues to perform this job today. With each whole-genome sequence publication of a new species — animal, plant, fungus, protist, eubacteria, archaebacteria, or virus — Nelson, by means of BLAST-searches, adds (and gives a name to) each new P450 gene in the CYP superfamily. The total number now exceeds 55,000 and is increasing every week.

Independently from Nebert’s vision, Margaret O Dayhoff — bioinformatics professor at Georgetown University Medical Center, and founder of Protein Identification Resource (PIR) — had a vision of amino-acid alignment and divergence based on evolution.^[52] Subsequently, the Universal Protein Resource (UniProt) is the world’s most comprehensive catalog of information on proteins; this central repository of protein sequence and function^[53] was created by joining the information contained in Swiss-Prot, TrEMBL, and PIR. Unfortunately, Dayhoff died in 1983 of a heart attack at age 57, shortly before Nebert had planned to meet with her.

Discovery of Slc39a8, encoding ZIP8 divalent cation transporter

“Forward genetics” is an experimental approach that starts with a phenotype (trait) and then seeks to find the gene (or genetic basis) causing that trait. On the other hand, “reverse genetics” begins with the gene (or genetic locus) and searches for phenotype(s) caused by that DNA sequence. “AHH inducibility by PAHs and dioxin,” as described above, is an example of an initial observation (phenotype) by which Nebert then used forward genetics (including the power of AHR-sensitive and AHR-resistant inbred mouse strains) to identify CYP1A1 regulation by AHR.

“Resistance to cadmium (Cd²⁺)-caused testicular necrosis” was shown by Benjamin A Taylor to segregate as an autosomal recessive trait.^[54] As with Nebert’s search for the Ahr locus, Taylor’s study represented forward genetics — using the power of Cd-sensitive and Cd-resistant inbred mouse strains. Taylor named the “cadmium resistance” (Cdm) locus, and, via a three-point cross, he placed the Cdm locus between two previously mapped genes, amylase-1 (Amy1) and varitint-waddler (Va).^[55] Taylor’s studies in the 1970s had always fascinated Nebert, who later directed his lab to corroborate and extend Taylor’s work; by studying two inbred mouse strains plus 26 recombinant inbred BXD/Ty lines plus using polymorphic satellite markers, the Cdm locus on chromosome 3 was narrowed from more than 24 centiMorgans (cM)^[55] to 0.64 cM.^[56]

The next step was to find the gene responsible for the Cd-responsive trait in the 0.64-cM region [which represented ≈4.96 megabases (Mb)]. Using SNV analysis of this region, plus studying two Cd-sensitive and two Cd-resistant inbred mouse strains along with the recombinant inbred BXD14/Ty line, Nebert with Timothy P Dalton demonstrated a 400-kilobase (kb) haplotype block associated with the Cd-induced toxicity phenotype.^[57] In this block was the Slc39a8 gene — encoding ZIP8, a member of the solute-carrier transporter superfamily; at that time the only homologous genes in the genome database were the putative zinc-responsive (ZRT)- iron-responsive transporter (IRT)-like Protein-8 (ZIP8) in plant and yeast. In mouse fetal fibroblast cultures, ZIP8 expression was associated with large increases in Cd²⁺ influx, accumulation, and toxicity. By in situ hybridization, ZIP8 mRNA was found to be prominent in testicular vascular endothelial cells of Cd-sensitive, but not Cd-resistant, strains of mice.^[57] ZIP8 expression was subsequently found to be highest in kidney, lung, testis, and ubiquitously expressed to varying degrees throughout the mouse.^[58]

A transgenic mouse line was created,^[58] which carried a bacterial artificial chromosome (BAC) with the Slc39a8 gene from the 129/SvJ Cd-sensitive mouse, inserted into the Cd-resistant C57BL/6J mouse. This BTZIP8-3 transgenic mouse was found to contain five Slc39a8 gene copies — three from the BAC, plus the two wild-type copies. ZIP8 mRNA and protein levels were shown to be located in the same tissues (but expressed ≈2½-times greater) in BTZIP8-3, compared with wild-type mice. Cd treatment reversed the Cd-resistance trait, seen in nontransgenic littermates, to Cd sensitivity in BTZIP8-3 mice; reversal of the testicular necrosis phenotype thus confirmed that the Slc39a8 gene is unequivocally the Cdm locus.^[58]

Nebert with Lei He then used stable retroviral infection of the ZIP8 cDNA in mouse fetal fibroblast cultures (rvZIP8 cells) to study divalent cation uptake kinetics and Km values; it was concluded^[59] that Mn²⁺, more than Zn²⁺, is the best physiological substrate for ZIP8. Fe²⁺ and Co²⁺ have also been suggested as ZIP8 substrates.^[60] In ZIP8-expressing Xenopus oocyte cultures, electrogenicity studies revealed an influx of two HCO₃^– anions per one Zn²⁺ (or Mn²⁺ or Cd²⁺) cation, i.e. an M²⁺/(HCO₃^–)₂ electroneutral complex.^[61] More recently, selenite (HSeO₃^–) — as a form of selenium that is taken up by cells — was shown to require Zn²⁺ and HCO₃^– and be transported by ZIP8; thus, Zn²⁺/(HCO₃^–)(HSeO₃^–) was proposed as the most likely electroneutral complex.^[62] The ZIP8 eight-transmembrane protein was also found to be largely internalized during Zn²⁺ treatment, as well as at homeostasis, but moves predominantly to the cell-surface membrane (via trafficking) under conditions of Zn²⁺ depletion in culture medium.^[61]

Again, fascinated by evolution, Nebert examined homology of amino-acid sequences among the Slc39a gene subfamily of 14 members in mouse — and found that Slc39a14 was most closely evolutionarily related to Slc39a8. The Nebert lab subsequently cloned and characterized the Slc39a14 gene. ZIP14 exhibits similar transporter properties to ZIP8, but tissue specificity of ZIP14 differs from that of ZIP8.^[63][64] Alignment of human and mouse SLC39A members showed a very high degree of evolutionary conservation between each ortholog.^[64] This finding strongly suggests these SLC39A genes have existed for at least the last 80 million years, and therefore are likely to be critical to fundamental life processes — including pivotal functions during early embryogenesis.

Previous studies had found that ZIP8 is expressed in gastrula^[65] and in visceral endoderm^[66] at GD7.5. In fact, ZIP8 has been proposed to be used as a potential indicator of cell differentiation (self-renewal-related signaling) in embryonic stem cells.^[67] Learning this, Nebert postulated that a Slc39a8(–/–) global knockout would likely be very early embryolethal. Nebert, with Lei He and Tim Dalton, confirmed this was the case.^[68]

During the process of attempting to create the global knockout, an intriguing “knockdown” Slc39a8(neo/neo) mouse was produced.^[69] This hypomorph, which expresses ZIP8 mRNA and protein levels ≈15% of that in all wild-type tissues examined, is viable until at least GD16.5 with some pups surviving until postnatal day 1. Here, then, was an experimental model that provided a window of time for studying ZIP8 functions in placenta, yolk sac, and fetal tissues during in utero growth [Fig. 3].

The Slc39a8(neo) allele was found to be associated with diminished Zn²⁺, Mn²⁺ and Fe²⁺ in mouse fetal fibroblast and liver-derived cultures. Levels of these cations were also decreased in several tissues of Slc39a8(neo/neo) newborns.^[69] Slc39a8(neo/neo) homozygotes — from GD11.5 until death — are extremely pale, and stunted in growth with hypomorphic limbs.

Additional abnormalities include severely hypoplastic spleen and substantially reduced size of liver, kidney, lung, and brain (cerebrum and cerebellum). Histologically, Slc39a8(neo/neo) neonates show decreased numbers of hematopoietic islands in yolk sac and liver. Low hemoglobin, hematocrit, red cell count, serum iron, and total iron-binding capacity confirmed a severe anemia.^[69]

In an attempt to explain the Slc39a8(neo/neo) phenotypic pleiotropy, Nebert with Jing Chen carried out bioinformatics analysis of the transcriptome in GD13.5 yolk sac and placenta, and in GD16.5 liver, kidney, lung, heart and cerebellum — comparing Slc39a8(neo/neo) with Slc39a8(+/+) wild-type.^[70] Based on transcription factor (TF) profiles and a search for enriched TF-binding sites, numerous genes encoding zinc-fingers and other TFs associated with hematopoietic stem cell functions were identified. It was concluded that in Slc39a8(neo/neo) mice, deficient ZIP8-mediated divalent cation transport — predominantly in yolk sac — affects zinc-finger protein TFs (such as GATA) and other TFs interacting with GATA proteins (e.g.. TAL1). These RNA-seq data^[70] strongly support the in utero phenotypes of dysmorphogenesis, dysregulated hematopoietic stem cell fate, and anemia seen in Slc39a8(neo/neo) mice.^[69]

The SLC39A8 gene was originally discovered in human monocytes,^[71] but was given a trivial name. ZIP8 function was shown in human lung and other cell cultures to protect against inflammation.^{[72][73][74][75]} Furthermore, increasing numbers of genome-wide association studies (GWAS) have identified human SLC39A8 variants correlated with clinical phenotypes: heart disease and blood pressure regulation,^{[76][77][78][79][80][81][82]} schizophrenia,^[83][84] osteoarthritis,^[85][86] Crohn’s disease,^[87] and retinal iron accumulation.^[88] SLC39A8 variants were also associated with congenital deformed skull, cerebellar atrophy, profound psychomotor retardation, severe seizures, short limbs, and hearing loss defects.^[89][90] ZIP8 deficiency was found to impair Mn²⁺-dependent enzyme function, which severely affects posttranslational glycosylation.^[89][90][91] This finding might explain many of the ZIP8-mediated pleiotropic clinical effects. The updated “SLC39A8 complete story”, as of summer 2019, was reviewed in depth.^[92]

Legacy

Two independent forward genetics studies — serendipitously chosen by Nebert and spanning a career of more than five decades — began with two intriguing phenotypes: the first was AHH induction by PAHs and dioxin; the second was cadmium-induced testicular necrosis. Both projects led to discovery of genes (AHR-regulation of CYP1A1; SLC39A8 coding for a divalent cation transporter, respectively) that are expressed in mammalian embryonic stem cells. And, in both cases, these genes are pivotal in critical life processes, i.e. disruption of either gene leads to many serious clinical disorders and/or embryolethality. Nebert emphasized to all his trainees to “focus on creative research, think outside the box.” During his entire career which included more than 100 grad student, postdoc and other trainees — every one of Nebert’s projects has revolved around the central theme of gene-environment interactions. Most of this research would never have been accomplished, without the hands of these trainees and valuable discussions with many dozens of colleagues over the years.

Marriage and family

Nebert was married to Myrna Sisk (1960-1974) and Kathleen Dixon, PhD (1981-1995). In 2000 he married Lucia Jorge Fung, PhD — who, at the time, was Chair of the Department of Medicinal Chemistry & Pharmacognosy, School of Pharmacy, at the University of Panama in the Republic of Panama. Professor Nebert has six children: Douglas Daniel (1962-2014); Dietrich Andrew (1967- ); Rosemarie Dixon (Korean adoption, 1983- ); Rebecca Frances (1984- ); David Porter (1986- ); and Lucas Daniel (1986- ); plus six grandchildren.

References

• Nebert, D. W.; Gelboin, H. V. (1968-12-10). “Substrate-inducible microsomal aryl hydroxylase in mammalian cell culture. I. Assay and properties of induced enzyme”. The Journal of Biological Chemistry. 243 (23): 6242–6249. ISSN 0021-9258. PMID 4387094.
•  
• Nebert, D. W.; Gelboin, H. V. (1968-12-10). “Substrate-inducible microsomal aryl hydroxylase in mammalian cell culture. II. Cellular responses during enzyme induction”. The Journal of Biological Chemistry. 243 (23): 6250–6261. ISSN 0021-9258. PMID 5723465.
•  
• Nebert, D. W. (1970-02-10). “Microsomal cytochromes b5 and P450 during induction of aryl hydrocarbon hydroxylase activity in mammalian cell culture”. The Journal of Biological Chemistry. 245 (3): 519–527. ISSN 0021-9258. PMID 4391690.
•  
• Nelson, D. R.; Koymans, L.; Kamataki, T.; Stegeman, J. J.; Feyereisen, R.; Waxman, D. J.; Waterman, M. R.; Gotoh, O.; Coon, M. J. (February 1996). “P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature”. Pharmacogenetics. 6 (1): 1–42. doi:10.1097/00008571-199602000-00002. ISSN 0960-314X. PMID 8845856.
•  
• Poland, A. P.; Glover, E.; Robinson, J. R.; Nebert, D. W. (1974-09-10). “Genetic expression of aryl hydrocarbon hydroxylase activity. Induction of monooxygenase activities and cytochrome P1-450 formation by 2,3,7,8-tetrachlorodibenzo-p-dioxin in mice genetically “nonresponsive” to other aromatic hydrocarbons”. The Journal of Biological Chemistry. 249 (17): 5599–5606. ISSN 0021-9258. PMID 4370044.
•  
• Nebert, D. W.; Robinson, J. R.; Niwa, A.; Kumaki, K.; Poland, A. P. (April 1975). “Genetic expression of aryl hydrocarbon hydroxylase activity in the mouse”. Journal of Cellular Physiology. 85 (2 Pt 2 Suppl 1): 393–414. doi:10.1002/jcp.1040850407. ISSN 0021-9541. PMID 1091656.
•  
• Poland, A.; Glover, E.; Kende, A. S. (1976-08-25). “Stereospecific, high affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin by hepatic cytosol. Evidence that the binding species is receptor for induction of aryl hydrocarbon hydroxylase”. The Journal of Biological Chemistry. 251 (16): 4936–4946. ISSN 0021-9258. PMID 956169.
•  
• Burbach, K. M.; Poland, A.; Bradfield, C. A. (1992-09-01). “Cloning of the Ah-receptor cDNA reveals a distinctive ligand-activated transcription factor”. Proceedings of the National Academy of Sciences of the United States of America. 89 (17): 8185–8189. Bibcode:1992PNAS…89.8185B. doi:10.1073/pnas.89.17.8185. ISSN 0027-8424. PMC 49882. PMID 1325649.
•  
• Le Beau, M. M.; Carver, L. A.; Espinosa, R.; Schmidt, J. V.; Bradfield, C. A. (1994). “Chromosomal localization of the human AHR locus encoding the structural gene for the Ah receptor to 7p21–>p15”. Cytogenetics and Cell Genetics. 66 (3): 172–176. doi:10.1159/000133694. ISSN 0301-0171. PMID 8125016.
•  
• Hahn, Mark E.; Karchner, Sibel I.; Evans, Brad R.; Franks, Diana G.; Merson, Rebeka R.; Lapseritis, Joy M. (2006-09-01). “Unexpected diversity of aryl hydrocarbon receptors in non-mammalian vertebrates: insights from comparative genomics”. Journal of Experimental Zoology Part A: Comparative Experimental Biology. 305 (9): 693–706. doi:10.1002/jez.a.323. ISSN 1548-8969. PMID 16902966.
•  
• Kewley, Robyn J.; Whitelaw, Murray L.; Chapman-Smith, Anne (February 2004). “The mammalian basic helix-loop-helix/PAS family of transcriptional regulators”. The International Journal of Biochemistry & Cell Biology. 36 (2): 189–204. doi:10.1016/S1357-2725(03)00211-5. ISSN 1357-2725. PMID 14643885.
•  
• Nebert, Daniel W. (July 2017). “Aryl hydrocarbon receptor (AHR): “pioneer member” of the basic-helix/loop/helix per-Arnt-sim (bHLH/PAS) family of “sensors” of foreign and endogenous signals”. Progress in Lipid Research. 67: 38–57. doi:10.1016/j.plipres.2017.06.001. ISSN 1873-2194. PMC 5568781. PMID 28606467.
•  
• Konopka, R. J.; Benzer, S. (September 1971). “Clock mutants of Drosophila melanogaster”. Proceedings of the National Academy of Sciences of the United States of America. 68 (9): 2112–2116. Bibcode:1971PNAS…68.2112K. doi:10.1073/pnas.68.9.2112. ISSN 0027-8424. PMC 389363. PMID 5002428.
•  
• Crews, S. T.; Thomas, J. B.; Goodman, C. S. (1988-01-15). “The Drosophila single-minded gene encodes a nuclear protein with sequence similarity to the per gene product”. Cell. 52 (1): 143–151. doi:10.1016/0092-8674(88)90538-7. ISSN 0092-8674. PMID 3345560.
•  
• Hoffman, E. C.; Reyes, H.; Chu, F. F.; Sander, F.; Conley, L. H.; Brooks, B. A.; Hankinson, O. (1991-05-17). “Cloning of a factor required for activity of the Ah (dioxin) receptor”. Science. 252 (5008): 954–958. Bibcode:1991Sci…252..954H. doi:10.1126/science.1852076. ISSN 0036-8075. PMID 1852076.
•  
• Nambu, J. R.; Lewis, J. O.; Wharton, K. A.; Crews, S. T. (1991-12-20). “The Drosophila single-minded gene encodes a helix-loop-helix protein that acts as a master regulator of CNS midline development”. Cell. 67 (6): 1157–1167. doi:10.1016/0092-8674(91)90292-7. ISSN 0092-8674. PMID 1760843.
•  
• Nebert, D. W.; Eisen, H. J.; Negishi, M.; Lang, M. A.; Hjelmeland, L. M.; Okey, A. B. (1981). “Genetic mechanisms controlling the induction of polysubstrate monooxygenase (P-450) activities”. Annual Review of Pharmacology and Toxicology. 21: 431–462. doi:10.1146/annurev.pa.21.040181.002243. ISSN 0362-1642. PMID 7016012.
•  
• Bigelow, S. W.; Collins, A. C.; Nebert, D. W. (1989-10-15). “Selective mouse breeding for short ethanol sleep time has led to high levels of hepatic aromatic hydrocarbon (Ah) receptor”. Biochemical Pharmacology. 38 (20): 3565–3572. doi:10.1016/0006-2952(89)90129-9. ISSN 0006-2952. PMID 2554926.
•  
• Nebert, D. W. (1994). “Drug metabolism and signal transduction: possible role of Ah receptor and arachidonic acid cascade in protection from ethanol toxicity”. EXS. 71: 231–240. doi:10.1007/978-3-0348-7330-7_23. ISBN 978-3-0348-7332-1. ISSN 1023-294X. PMID 8032154.
•  
• Rifkind, A. B.; Muschick, H. (June 1983). “Benoxaprofen suppression of polychlorinated biphenyl toxicity without alteration of mixed function oxidase function”. Nature. 303 (5917): 524–526. Bibcode:1983Natur.303..524R. doi:10.1038/303524a0. ISSN 0028-0836. PMID 6406903.
•  
• Divanovic, Senad; Dalli, Jesmond; Jorge-Nebert, Lucia F.; Flick, Leah M.; Gálvez-Peralta, Marina; Boespflug, Nicholas D.; Stankiewicz, Traci E.; Fitzgerald, Jonathan M.; Somarathna, Maheshika (2013-09-15). “Contributions of the three CYP1 monooxygenases to pro-inflammatory and inflammation-resolution lipid mediator pathways”. Journal of Immunology. 191 (6): 3347–3357. doi:10.4049/jimmunol.1300699. ISSN 1550-6606. PMC 3810452. PMID 23956430.
•  
• Nebert, D. W. (1989). “The Ah locus: genetic differences in toxicity, cancer, mutation, and birth defects”. Critical Reviews in Toxicology. 20 (3): 153–174. doi:10.3109/10408448909017908. ISSN 1040-8444. PMID 2558673.
•  
• Nebert, D. W.; Roe, A. L.; Dieter, M. Z.; Solis, W. A.; Yang, Y.; Dalton, T. P. (2000-01-01). “Role of the aromatic hydrocarbon receptor and [Ah] gene battery in the oxidative stress response, cell cycle control, and apoptosis”. Biochemical Pharmacology. 59 (1): 65–85. doi:10.1016/S0006-2952(99)00310-X. ISSN 0006-2952. PMID 10605936.
•  
• Gonzalez, F. J.; Kimura, S.; Nebert, D. W. (1985-04-25). “Comparison of the flanking regions and introns of the mouse 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible cytochrome P1-450 and P3-450 genes”. The Journal of Biological Chemistry. 260 (8): 5040–5049. ISSN 0021-9258. PMID 3988744.
•  
• Jaiswal, A. K.; Gonzalez, F. J.; Nebert, D. W. (1985-06-25). “Human P1-450 gene sequence and correlation of mRNA with genetic differences in benzo[a]pyrene metabolism”. Nucleic Acids Research. 13 (12): 4503–4520. doi:10.1093/nar/13.12.4503. ISSN 0305-1048. PMC 321802. PMID 2989797.
•  
• Ikeya, K.; Jaiswal, A. K.; Owens, R. A.; Jones, J. E.; Nebert, D. W.; Kimura, S. (September 1989). “Human CYP1A2: sequence, gene structure, comparison with the mouse and rat orthologous gene, and differences in liver 1A2 mRNA expression”. Molecular Endocrinology (Baltimore, Md.). 3 (9): 1399–1408. doi:10.1210/mend-3-9-1399. ISSN 0888-8809. PMID 2575218.
•  
• Fernandez-Salguero, P.; Pineau, T.; Hilbert, D. M.; McPhail, T.; Lee, S. S.; Kimura, S.; Nebert, D. W.; Rudikoff, S.; Ward, J. M. (1995-05-05). “Immune system impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor”. Science. 268 (5211): 722–726. Bibcode:1995Sci…268..722F. doi:10.1126/science.7732381. ISSN 0036-8075. PMID 7732381.
•  
• Liang, H. C.; Li, H.; McKinnon, R. A.; Duffy, J. J.; Potter, S. S.; Puga, A.; Nebert, D. W. (1996-02-20). “Cyp1a2(-/-) null mutant mice develop normally but show deficient drug metabolism”. Proceedings of the National Academy of Sciences of the United States of America. 93 (4): 1671–1676. Bibcode:1996PNAS…93.1671L. doi:10.1073/pnas.93.4.1671. ISSN 0027-8424. PMC 40000. PMID 8643688.
•  
• Uno, S.; Dalton, T. P.; Shertzer, H. G.; Genter, M. B.; Warshawsky, D.; Talaska, G.; Nebert, D. W. (2001-12-21). “Benzo[a]pyrene-induced toxicity: paradoxical protection in Cyp1a1(-/-) knockout mice having increased hepatic BaP-DNA adduct levels”. Biochemical and Biophysical Research Communications. 289 (5): 1049–1056. doi:10.1006/bbrc.2001.6110. ISSN 0006-291X. PMID 11741297.
•  
• Uno, Shigeyuki; Wang, Bin; Shertzer, Howard G.; Nebert, Daniel W.; Dalton, Timothy P. (2003-12-12). “Balancer-Cre transgenic mouse germ cells direct the incomplete resolution of a tri-loxP-targeted Cyp1a1 allele, producing a conditional knockout allele”. Biochemical and Biophysical Research Communications. 312 (2): 494–499. doi:10.1016/j.bbrc.2003.10.145. ISSN 0006-291X. PMID 14637164.
•  
• Shi, Zhanquan; Dragin, Nadine; Gálvez-Peralta, Marina; Jorge-Nebert, Lucia F.; Miller, Marian L.; Wang, Bin; Nebert, Daniel W. (July 2010). “Organ-specific roles of CYP1A1 during detoxication of dietary benzo[a]pyrene”. Molecular Pharmacology. 78 (1): 46–57. doi:10.1124/mol.110.063438. ISSN 1521-0111. PMC 2912055. PMID 20371670.
•  
• Uno, Shigeyuki; Dalton, Timothy P.; Derkenne, Sandrine; Curran, Christine P.; Miller, Marian L.; Shertzer, Howard G.; Nebert, Daniel W. (May 2004). “Oral exposure to benzo[a]pyrene in the mouse: detoxication by inducible cytochrome P450 is more important than metabolic activation”. Molecular Pharmacology. 65 (5): 1225–1237. doi:10.1124/mol.65.5.1225. ISSN 0026-895X. PMID 15102951. S2CID 24627183.
•  
• Uno, Shigeyuki; Dalton, Timothy P.; Dragin, Nadine; Curran, Christine P.; Derkenne, Sandrine; Miller, Marian L.; Shertzer, Howard G.; Gonzalez, Frank J.; Nebert, Daniel W. (April 2006). “Oral benzo[a]pyrene in Cyp1 knockout mouse lines: CYP1A1 important in detoxication, CYP1B1 metabolism required for immune damage independent of total-body burden and clearance rate”. Molecular Pharmacology. 69 (4): 1103–1114. doi:10.1124/mol.105.021501. ISSN 0026-895X. PMID 16377763.
•  
• Dragin, Nadine; Shi, Zhanquan; Madan, Rajat; Karp, Christopher L.; Sartor, Maureen A.; Chen, Chi; Gonzalez, Frank J.; Nebert, Daniel W. (June 2008). “Phenotype of the Cyp1a1/1a2/1b1-/- triple-knockout mouse”. Molecular Pharmacology. 73 (6): 1844–1856. doi:10.1124/mol.108.045658. ISSN 1521-0111. PMC 2735564. PMID 18372398.
•  
• Nebert, Daniel W.; Karp, Christopher L. (2008-12-26). “Endogenous functions of the aryl hydrocarbon receptor (AHR): intersection of cytochrome P450 1 (CYP1)-metabolized eicosanoids and AHR biology”. The Journal of Biological Chemistry. 283 (52): 36061–36065. doi:10.1074/jbc.R800053200. ISSN 0021-9258. PMC 2606007. PMID 18713746.
•  
• Nebert, Daniel W.; Shi, Zhanquan; Gálvez-Peralta, Marina; Uno, Shigeyuki; Dragin, Nadine (September 2013). “Oral benzo[a]pyrene: understanding pharmacokinetics, detoxication, and consequences–Cyp1 knockout mouse lines as a paradigm”. Molecular Pharmacology. 84 (3): 304–313. doi:10.1124/mol.113.086637. ISSN 1521-0111. PMC 3876811. PMID 23761301.
•  
• Galloway, S. M.; Perry, P. E.; Meneses, J.; Nebert, D. W.; Pedersen, R. A. (June 1980). “Cultured mouse embryos metabolize benzo[a]pyrene during early gestation: genetic differences detectable by sister chromatid exchange”. Proceedings of the National Academy of Sciences of the United States of America. 77 (6): 3524–3528. Bibcode:1980PNAS…77.3524G. doi:10.1073/pnas.77.6.3524. ISSN 0027-8424. PMC 349649. PMID 6932035.
•  
• Dey, A.; Nebert, D. W. (1998-10-20). “Markedly increased constitutive CYP1A1 mRNA levels in the fertilized ovum of the mouse”. Biochemical and Biophysical Research Communications. 251 (2): 657–661. doi:10.1006/bbrc.1998.9519. ISSN 0006-291X. PMID 9792830.
•  
• Kärenlampi, S. O.; Eisen, H. J.; Hankinson, O.; Nebert, D. W. (1983-09-10). “Effects of cytochrome P1-450 inducers on the cell-surface receptors for epidermal growth factor, phorbol 12,13-dibutyrate, or insulin of cultured mouse hepatoma cells”. The Journal of Biological Chemistry. 258 (17): 10378–10383. ISSN 0021-9258. PMID 6309801.
•  
• Kimura, S.; Donovan, J. C.; Nebert, D. W. (1987). “Expression of the mouse P(1)450 gene during differentiation without foreign chemical stimulation”. Journal of Experimental Pathology. 3 (1): 61–74. ISSN 0730-8485. PMID 3681488.
•  
• Motulsky, A. G. (1957-10-19). “Drug reactions enzymes, and biochemical genetics”. Journal of the American Medical Association. 165 (7): 835–837. doi:10.1001/jama.1957.72980250010016. ISSN 0002-9955. PMID 13462859.
•  
• Vogel, Friedrich (1959). “Moderne Probleme der Humangenetik”. In Heilmeyer, L.; Schoen, R.; de Rudder, B. (eds.). Ergebnisse der Inneren Medizin und Kinderheilkunde. Ergebnisse der Inneren Medizin und Kinderheilkunde (in German). Springer Berlin Heidelberg. pp. 52–125. doi:10.1007/978-3-642-94744-5_2. ISBN 9783642947445.
•  
• Atlas, S. A.; Nebert, D. W. (March 1978). “Pharmacogenetics: a possible pragmatic perspective in neoplasm predictability”. Seminars in Oncology. 5 (1): 89–106. ISSN 0093-7754. PMID 345457.
•  
• Nebert, D. W. (October 1999). “Pharmacogenetics and pharmacogenomics: why is this relevant to the clinical geneticist?”. Clinical Genetics. 56 (4): 247–258. doi:10.1034/j.1399-0004.1999.560401.x. ISSN 0009-9163. PMID 10636440.
•  
• Nebert, Daniel W.; Jorge-Nebert, Lucia; Vesell, Elliot S. (2003). “Pharmacogenomics and “individualized drug therapy”: high expectations and disappointing achievements”. American Journal of Pharmacogenomics: Genomics-Related Research in Drug Development and Clinical Practice. 3 (6): 361–370. doi:10.2165/00129785-200303060-00002. ISSN 1175-2203. PMID 14672516.
•  
• Nebert, Daniel W.; Zhang, Ge; Vesell, Elliot S. (2008). “From human genetics and genomics to pharmacogenetics and pharmacogenomics: past lessons, future directions”. Drug Metabolism Reviews. 40 (2): 187–224. doi:10.1080/03602530801952864. ISSN 1097-9883. PMC 2752627. PMID 18464043.
•  
• Zhang, Ge; Nebert, Daniel W. (July 2017). “Personalized medicine: Genetic risk prediction of drug response”. Pharmacology & Therapeutics. 175: 75–90. doi:10.1016/j.pharmthera.2017.02.036. ISSN 1879-016X. PMC 5653378. PMID 28213088.
•  
• Elsevier. “Emery and Rimoin’s Principles and Practice of Medical Genetics – 6th Edition”. www.elsevier.com. Retrieved 2019-01-14.
•  
• Nebert, D. W.; Gonzalez, F. J. (1987). “P450 genes: structure, evolution, and regulation”. Annual Review of Biochemistry. 56: 945–993. doi:10.1146/annurev.bi.56.070187.004501. ISSN 0066-4154. PMID 3304150.
•  
• Nebert, D. W.; Adesnik, M.; Coon, M. J.; Estabrook, R. W.; Gonzalez, F. J.; Guengerich, F. P.; Gunsalus, I. C.; Johnson, E. F.; Kemper, B. (February 1987). “The P450 gene superfamily: recommended nomenclature”. DNA. 6 (1): 1–11. doi:10.1089/dna.1987.6.1. ISSN 0198-0238. PMID 3829886.
•  
• Nebert, D. W.; Nelson, D. R.; Adesnik, M.; Coon, M. J.; Estabrook, R. W.; Gonzalez, F. J.; Guengerich, F. P.; Gunsalus, I. C.; Johnson, E. F. (January 1989). “The P450 superfamily: updated listing of all genes and recommended nomenclature for the chromosomal loci”. DNA. 8 (1): 1–13. doi:10.1089/dna.1.1989.8.1. ISSN 0198-0238. PMID 2651058.
•  
• Hunt, L. T.; George, D. G.; Yeh, L. S.; Dayhoff, M. O. (1984). “Evolution of prokaryote and eukaryote lines inferred from sequence evidence”. Origins of Life. 14 (1–4): 657–664. Bibcode:1984OrLi…14..657H. doi:10.1007/BF00933718. ISSN 0302-1688. PMID 6087240.
•  
• The UniProt Consortium (January 2017). “UniProt: the universal protein knowledgebase”. Nucleic Acids Research. 45 (D1): D158–D169. doi:10.1093/nar/gkw1099. ISSN 1362-4962. PMC 5210571. PMID 27899622.
•  
• Taylor, B. A.; Heiniger, H. J.; Meier, H. (July 1973). “Genetic analysis of resistance to cadmium-induced testicular damage in mice”. Proceedings of the Society for Experimental Biology and Medicine (New York, N.Y.). 143 (3): 629–633. doi:10.3181/00379727-143-37380. ISSN 0037-9727. PMID 4719448.
•  
• Taylor, B. A. (November 1976). “Linkage of the cadmium resistance locus to loci on mouse chromosome 12”. The Journal of Heredity. 67 (6): 389–390. doi:10.1093/oxfordjournals.jhered.a108759. ISSN 0022-1503. PMID 1021597.
•  
• Dalton, T. P.; Miller, M. L.; Wu, X.; Menon, A.; Cianciolo, E.; McKinnon, R. A.; Smith, P. W.; Robinson, L. J.; Nebert, D. W. (March 2000). “Refining the mouse chromosomal location of Cdm, the major gene associated with susceptibility to cadmium-induced testicular necrosis”. Pharmacogenetics. 10 (2): 141–151. doi:10.1097/00008571-200003000-00006. ISSN 0960-314X. PMID 10762002.
•  
• Dalton, Timothy P.; He, Lei; Wang, Bin; Miller, Marian L.; Jin, Li; Stringer, Keith F.; Chang, Xiaoqing; Baxter, C. Stuart; Nebert, Daniel W. (2005-03-01). “Identification of mouse SLC39A8 as the transporter responsible for cadmium-induced toxicity in the testis”. Proceedings of the National Academy of Sciences of the United States of America. 102 (9): 3401–3406. Bibcode:2005PNAS..102.3401D. doi:10.1073/pnas.0406085102. ISSN 0027-8424. PMC 552906. PMID 15722412.
•  
• Wang, Bin; Schneider, Scott N.; Dragin, Nadine; Girijashanker, Kuppuswami; Dalton, Timothy P.; He, Lei; Miller, Marian L.; Stringer, Keith F.; Soleimani, Manoocher (April 2007). “Enhanced cadmium-induced testicular necrosis and renal proximal tubule damage caused by gene-dose increase in a Slc39a8-transgenic mouse line”. American Journal of Physiology. Cell Physiology. 292 (4): C1523–1535. doi:10.1152/ajpcell.00409.2006. ISSN 0363-6143. PMID 17108009. S2CID 22335871.
•  
• He, Lei; Girijashanker, Kuppuswami; Dalton, Timothy P.; Reed, Jodie; Li, Hong; Soleimani, Manoocher; Nebert, Daniel W. (July 2006). “ZIP8, member of the solute-carrier-39 (SLC39) metal-transporter family: characterization of transporter properties”. Molecular Pharmacology. 70 (1): 171–180. doi:10.1124/mol.106.024521. ISSN 0026-895X. PMID 16638970.
•  
• Wang, Chia-Yu; Jenkitkasemwong, Supak; Duarte, Stephanie; Sparkman, Brian K.; Shawki, Ali; Mackenzie, Bryan; Knutson, Mitchell D. (2012-10-05). “ZIP8 is an iron and zinc transporter whose cell-surface expression is up-regulated by cellular iron loading”. The Journal of Biological Chemistry. 287 (41): 34032–34043. doi:10.1074/jbc.M112.367284. ISSN 1083-351X. PMC 3464513. PMID 22898811.
•  
• Liu, Zhiwei; Li, Hong; Soleimani, Manoocher; Girijashanker, Kuppuswami; Reed, Jodie M.; He, Lei; Dalton, Timothy P.; Nebert, Daniel W. (2008-01-25). “Cd2+ versus Zn2+ uptake by the ZIP8 HCO3–dependent symporter: kinetics, electrogenicity and trafficking”. Biochemical and Biophysical Research Communications. 365 (4): 814–820. doi:10.1016/j.bbrc.2007.11.067. ISSN 1090-2104. PMC 2212618. PMID 18037372.
•  
• McDermott, Joseph R.; Geng, Xiangrong; Jiang, Lan; Gálvez-Peralta, Marina; Chen, Fei; Nebert, Daniel W.; Liu, Zijuan (2016-06-07). “Zinc- and bicarbonate-dependent ZIP8 transporter mediates selenite uptake”. Oncotarget. 7 (23): 35327–35340. doi:10.18632/oncotarget.9205. ISSN 1949-2553. PMC 5085232. PMID 27166256.
•  
• Girijashanker, Kuppuswami; He, Lei; Soleimani, Manoocher; Reed, Jodie M.; Li, Hong; Liu, Zhiwei; Wang, Bin; Dalton, Timothy P.; Nebert, Daniel W. (May 2008). “Slc39a14 gene encodes ZIP14, a metal/bicarbonate symporter: similarities to the ZIP8 transporter”. Molecular Pharmacology. 73 (5): 1413–1423. doi:10.1124/mol.107.043588. ISSN 1521-0111. PMC 2753210. PMID 18270315.
•  
• He, Lei; Wang, Bin; Hay, Everett B.; Nebert, Daniel W. (2009-08-01). “Discovery of ZIP transporters that participate in cadmium damage to testis and kidney”. Toxicology and Applied Pharmacology. 238 (3): 250–257. doi:10.1016/j.taap.2009.02.017. ISSN 1096-0333. PMC 2789294. PMID 19265717.
•  
• Harrison, S. M.; Dunwoodie, S. L.; Arkell, R. M.; Lehrach, H.; Beddington, R. S. (August 1995). “Isolation of novel tissue-specific genes from cDNA libraries representing the individual tissue constituents of the gastrulating mouse embryo”. Development. 121 (8): 2479–2489. ISSN 0950-1991. PMID 7671812.
•  
• Moore-Scott, Billie A.; Opoka, Robert; Lin, Suh-Chin J.; Kordich, Jennifer J.; Wells, James M. (July 2007). “Identification of molecular markers that are expressed in discrete anterior-posterior domains of the endoderm from the gastrula stage to mid-gestation”. Developmental Dynamics. 236 (7): 1997–2003. doi:10.1002/dvdy.21204. ISSN 1058-8388. PMID 17576135.
•  
• Zhu, Hao; Yang, He; Owen, Markus R. (December 2007). “Combined microarray analysis uncovers self-renewal related signaling in mouse embryonic stem cells”. Systems and Synthetic Biology. 1 (4): 171–181. doi:10.1007/s11693-008-9015-2. ISSN 1872-5325. PMC 2553326. PMID 19003442.
•  
• Wang, Bin; He, Lei; Dong, Hongbin; Dalton, Timothy P.; Nebert, Daniel W. (2011-07-01). “Generation of a Slc39a8 hypomorph mouse: markedly decreased ZIP8 Zn²⁺/(HCO₃⁻)₂ transporter expression”. Biochemical and Biophysical Research Communications. 410 (2): 289–294. doi:10.1016/j.bbrc.2011.05.134. ISSN 1090-2104. PMC 3136049. PMID 21658371.
•  
• Gálvez-Peralta, Marina; He, Lei; Jorge-Nebert, Lucia F.; Wang, Bin; Miller, Marian L.; Eppert, Bryan L.; Afton, Scott; Nebert, Daniel W. (2012). “ZIP8 zinc transporter: indispensable role for both multiple-organ organogenesis and hematopoiesis in utero”. PLOS ONE. 7 (5): e36055. Bibcode:2012PLoSO…736055G. doi:10.1371/journal.pone.0036055. ISSN 1932-6203. PMC 3341399. PMID 22563477.
•  
• Chen, Jing; Gálvez-Peralta, Marina; Zhang, Xiang; Deng, Jingyuan; Liu, Zijuan; Nebert, Daniel W. (2018-07-16). “In utero gene expression in the Slc39a8(neo/neo) knockdown mouse”. Scientific Reports. 8 (1): 10703. Bibcode:2018NatSR…810703C. doi:10.1038/s41598-018-29109-y. ISSN 2045-2322. PMC 6048144. PMID 30013175.
•  
• Begum, Nasim A.; Kobayashi, Mika; Moriwaki, Yasuhiro; Matsumoto, Misako; Toyoshima, Kumao; Seya, Tsukasa (December 2002). “Mycobacterium bovis BCG cell wall and lipopolysaccharide induce a novel gene, BIGM103, encoding a 7-TM protein: identification of a new protein family having Zn-transporter and Zn-metalloprotease signatures”. Genomics. 80 (6): 630–645. doi:10.1006/geno.2002.7000. ISSN 0888-7543. PMID 12504855.
•  
• Besecker, Beth; Bao, Shengying; Bohacova, Barbara; Papp, Audrey; Sadee, Wolfgang; Knoell, Daren L. (June 2008). “The human zinc transporter SLC39A8 (Zip8) is critical in zinc-mediated cytoprotection in lung epithelia”. American Journal of Physiology. Lung Cellular and Molecular Physiology. 294 (6): L1127–1136. doi:10.1152/ajplung.00057.2008. ISSN 1040-0605. PMID 18390834.
•  
• Liu, Ming-Jie; Bao, Shengying; Gálvez-Peralta, Marina; Pyle, Charlie J.; Rudawsky, Andrew C.; Pavlovicz, Ryan E.; Killilea, David W.; Li, Chenglong; Nebert, Daniel W.; Wewers, Martin D.; Knoell, Daren L. (2013-02-21). “ZIP8 regulates host defense through zinc-mediated inhibition of NF-κB”. Cell Reports. 3 (2): 386–400. doi:10.1016/j.celrep.2013.01.009. ISSN 2211-1247. PMC 3615478. PMID 23403290.
•  
• Pyle, Charlie J.; Akhter, Saife; Bao, ShengYing; Dodd, Claire E.; Schlesinger, Larry S.; Knoell, Daren L. (2017). “Zinc Modulates Endotoxin-Induced Human Macrophage Inflammation through ZIP8 Induction and C/EBPβ Inhibition”. PLOS ONE. 12 (1): e0169531. Bibcode:2017PLoSO..1269531P. doi:10.1371/journal.pone.0169531. ISSN 1932-6203. PMC 5215883. PMID 28056086.
•  
• Pyle, Charlie J.; Azad, Abul K.; Papp, Audrey C.; Sadee, Wolfgang; Knoell, Daren L.; Schlesinger, Larry S. (2017-11-09). “Elemental Ingredients in the Macrophage Cocktail: Role of ZIP8 in Host Response to Mycobacterium tuberculosis”. International Journal of Molecular Sciences. 18 (11): E2375. doi:10.3390/ijms18112375. ISSN 1422-0067. PMC 5713344. PMID 29120360.
•  
• Waterworth, Dawn M.; Ricketts, Sally L.; Song, Kijoung; Chen, Li; Zhao, Jing Hua; Ripatti, Samuli; Aulchenko, Yurii S.; Zhang, Weihua; Yuan, Xin (November 2010). “Genetic variants influencing circulating lipid levels and risk of coronary artery disease”. Arteriosclerosis, Thrombosis, and Vascular Biology. 30 (11): 2264–2276. doi:10.1161/ATVBAHA.109.201020. ISSN 1524-4636. PMC 3891568. PMID 20864672.
•  
• International Consortium for Blood Pressure Genome-Wide Association Studies; Ehret, Georg B.; Munroe, Patricia B.; Rice, Kenneth M.; Bochud, Murielle; Johnson, Andrew D.; Chasman, Daniel I.; Smith, Albert V.; Tobin, Martin D. (2011-09-11). “Genetic variants in novel pathways influence blood pressure and cardiovascular disease risk”. Nature. 478 (7367): 103–109. Bibcode:2011Natur.478..103T. doi:10.1038/nature10405. ISSN 1476-4687. PMC 3340926. PMID 21909115.
•  
• Ehret, Georg B.; Ferreira, Teresa; Chasman, Daniel I.; Jackson, Anne U.; Schmidt, Ellen M.; Johnson, Toby; Thorleifsson, Gudmar; Luan, Jian’an; Donnelly, Lousie A. (October 2016). “The genetics of blood pressure regulation and its target organs from association studies in 342,415 individuals”. Nature Genetics. 48 (10): 1171–1184. doi:10.1038/ng.3667. ISSN 1546-1718. PMC 5042863. PMID 27618452.
•  
• Teslovich, Tanya M.; Musunuru, Kiran; Smith, Albert V.; Edmondson, Andrew C.; Stylianou, Ioannis M.; Koseki, Masahiro; Pirruccello, James P.; Ripatti, Samuli; Chasman, Daniel I. (2010-08-05). “Biological, clinical and population relevance of 95 loci for blood lipids”. Nature. 466 (7307): 707–713. Bibcode:2010Natur.466..707T. doi:10.1038/nature09270. ISSN 1476-4687. PMC 3039276. PMID 20686565.
•  
• Willer, Cristen J.; Schmidt, Ellen M.; Sengupta, Sebanti; Peloso, Gina M.; Gustafsson, Stefan; Kanoni, Stavroula; Ganna, Andrea; Chen, Jin; Buchkovich, Martin L. (November 2013). “Discovery and refinement of loci associated with lipid levels”. Nature Genetics. 45 (11): 1274–1283. doi:10.1038/ng.2797. ISSN 1546-1718. PMC 3838666. PMID 24097068.
•  
• Verdugo, Ricardo A.; Zeller, Tanja; Rotival, Maxime; Wild, Philipp S.; Münzel, Thomas; Lackner, Karl J.; Weidmann, Henri; Ninio, Ewa; Trégouët, David-Alexandre (2013). “Graphical modeling of gene expression in monocytes suggests molecular mechanisms explaining increased atherosclerosis in smokers”. PLOS ONE. 8 (1): e50888. Bibcode:2013PLoSO…850888V. doi:10.1371/journal.pone.0050888. ISSN 1932-6203. PMC 3553098. PMID 23372645.
•  
• Johansson, Åsa; Eriksson, Niclas; Lindholm, Daniel; Varenhorst, Christoph; James, Stefan; Syvänen, Ann-Christine; Axelsson, Tomas; Siegbahn, Agneta; Barratt, Bryan J. (2016-04-01). “Genome-wide association and Mendelian randomization study of NT-proBNP in patients with acute coronary syndrome”. Human Molecular Genetics. 25 (7): 1447–1456. doi:10.1093/hmg/ddw012. ISSN 1460-2083. PMID 26908625.
•  
• Carrera, Noa; Arrojo, Manuel; Sanjuán, Julio; Ramos-Ríos, Ramón; Paz, Eduardo; Suárez-Rama, Jose J.; Páramo, Mario; Agra, Santiago; Brenlla, Julio (2012-01-15). “Association study of nonsynonymous single nucleotide polymorphisms in schizophrenia”. Biological Psychiatry. 71 (2): 169–177. doi:10.1016/j.biopsych.2011.09.032. ISSN 1873-2402. PMID 22078303.
•  
• Schizophrenia Working Group of the Psychiatric Genomics Consortium (2014-07-24). “Biological insights from 108 schizophrenia-associated genetic loci”. Nature. 511 (7510): 421–427. Bibcode:2014Natur.511..421S. doi:10.1038/nature13595. ISSN 1476-4687. PMC 4112379. PMID 25056061.
•  
• Song, Jinsoo; Kim, Dongkyun; Lee, Chang Hoon; Lee, Myeung Su; Chun, Churl-Hong; Jin, Eun-Jung (2013-05-20). “MicroRNA-488 regulates zinc transporter SLC39A8/ZIP8 during pathogenesis of osteoarthritis”. Journal of Biomedical Science. 20: 31. doi:10.1186/1423-0127-20-31. ISSN 1423-0127. PMC 3706240. PMID 23688035.
•  
• Kim, Jin-Hong; Jeon, Jimin; Shin, Minhee; Won, Yoonkyung; Lee, Minju; Kwak, Ji-Sun; Lee, Gyuseok; Rhee, Jinseol; Ryu, Je-Hwang (2014-02-13). “Regulation of the catabolic cascade in osteoarthritis by the zinc-ZIP8-MTF1 axis”. Cell. 156 (4): 730–743. doi:10.1016/j.cell.2014.01.007. ISSN 1097-4172. PMID 24529376.
•  
• Li, Dalin; Achkar, Jean-Paul; Haritunians, Talin; Jacobs, Jonathan P.; Hui, Ken Y.; D’Amato, Mauro; Brand, Stephan; Radford-Smith, Graham; Halfvarson, Jonas (October 2016). “A Pleiotropic Missense Variant in SLC39A8 Is Associated With Crohn’s Disease and Human Gut Microbiome Composition”. Gastroenterology. 151 (4): 724–732. doi:10.1053/j.gastro.2016.06.051. ISSN 1528-0012. PMC 5037008. PMID 27492617.
•  
• Baumann, Bailey; Sterling, Jacob; Song, Ying; Song, Delu; Fruttiger, Marcus; Gillies, Mark; Shen, Weiyong; Dunaief, Joshua L. (August 2017). “Conditional Müller Cell Ablation Leads to Retinal Iron Accumulation”. Investigative Ophthalmology & Visual Science. 58 (10): 4223–4234. doi:10.1167/iovs.17-21743. ISSN 1552-5783. PMC 5574447. PMID 28846772.
•  
• Boycott, Kym M.; Beaulieu, Chandree L.; Kernohan, Kristin D.; Gebril, Ola H.; Mhanni, Aziz; Chudley, Albert E.; Redl, David; Qin, Wen; Hampson, Sarah (2015-12-03). “Autosomal-Recessive Intellectual Disability with Cerebellar Atrophy Syndrome Caused by Mutation of the Manganese and Zinc Transporter Gene SLC39A8”. American Journal of Human Genetics. 97 (6): 886–893. doi:10.1016/j.ajhg.2015.11.002. ISSN 1537-6605. PMC 4678439. PMID 26637978.
•  
• Park, Julien H.; Hogrebe, Max; Grüneberg, Marianne; DuChesne, Ingrid; von der Heiden, Ava L.; Reunert, Janine; Schlingmann, Karl P.; Boycott, Kym M.; Beaulieu, Chandree L. (2015-12-03). “SLC39A8 Deficiency: A Disorder of Manganese Transport and Glycosylation”. American Journal of Human Genetics. 97 (6): 894–903. doi:10.1016/j.ajhg.2015.11.003. ISSN 1537-6605. PMC 4678430. PMID 26637979.
•  
• Riley, Lisa G.; Cowley, Mark J.; Gayevskiy, Velimir; Roscioli, Tony; Thorburn, David R.; Prelog, Kristina; Bahlo, Melanie; Sue, Carolyn M.; Balasubramaniam, Shanti (March 2017). “A SLC39A8 variant causes manganese deficiency, and glycosylation and mitochondrial disorders”. Journal of Inherited Metabolic Disease. 40 (2): 261–269. doi:10.1007/s10545-016-0010-6. ISSN 1573-2665. PMID 27995398.
•  

Nebert, D.W.; Liu, Z. (Sep 2019). “SLC39A8 gene encoding a metal ion transporter: discovery and bench to bedside”. Human Genomics. 13 (Suppl 1): 51. doi:10.1186/s40246-019-0233-3. PMC 6744627. PMID 31521203