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Previous large-scale vaccination clinics have been successful before the coronavirus disease 2019 (COVID-19) pandemic; however, owing to the strict storage requirements and pharmaceutical preparation needed for the COVID-19 vaccines, careful thought and planning were necessary to successfully deploy these clinics immediately after vaccine availability. The focus of this manuscript is to describe the development and implementation of COVID-19 vaccination clinics in a large public university, using professionals from within and outside of its health sciences schools.The primary objective of this project was to (1) implement COVID-19 vaccination clinics for university faculty, staff, students, and community members. Additional objectives of the clinics were to (2) actively incorporate pharmacy, nursing, and medical students into the clinic workflow; (3) promote interprofessional collaboration among faculty and students; and (4) assess patient satisfaction.The School of Pharmacy faculty, in conjunction with the Office of Strategic Initiatives, planned and coordinated COVID-19 vaccination clinics from December 2020 to July 2021. Students and faculty from schools of pharmacy, nursing, and medicine were used. COVID-19 vaccinations were offered to university faculty, staff, and students and community members based on the Centers for Disease Control and Prevention priority groups. The clinic processes were designed such that they could be scaled from 100 to 2,000 participants per day.The School of Pharmacy led approach was adjustable depending on the number of patients, continuously monitored and adaptable. The importance of pharmacists as part of the interprofessional health care team was exemplified by faculty and students involved.All patients receiving COVID-19 vaccinations at the clinics were e-mailed anonymous surveys for assessment of the quality of the vaccination encounter after completion of their primary vaccine series.More than 15,000 COVID-19 vaccinations were provided through the clinics from December 2020 to July 2021. Professional staffing totaled 3352 hours for the 48 clinics. Thirty-eight percent of the vaccinated patients responded to the clinic satisfaction survey with predominately excellent ratings.COVID-19 vaccination clinics can be successfully planned and implemented in a scalable fashion in a large university setting using an interprofessional team approach.

Pharmacy students experience high levels of perceived stress. Data regarding the impact of curricular revision on students' stress level are lacking. The primary objective of this study was to compare perceived stress, academic self-concept, and coping strategies between pharmacy students prior to and following a curricular revision. Secondary objectives included determining university resources used by students to deal with stress.Students in the first, second, and third years of the pharmacy curriculum were asked to complete a survey, including the 14-item Perceived Stress Scale (PSS-14), Brief COPE, and Academic Self-Concept Scale (ASCS), and questions regarding use of university resources. Responses to the PSS-14, Brief COPE, and ASCS were compared to a student cohort prior to the curricular revision.Perceived stress was reduced to a small, statistically significant degree following a curricular revision. In both cohorts, increased stress was statistically significantly correlated with decreased academic self-concept. Students reported increased use of self-distraction, along with decreased use of active coping, substance abuse, and planning, as coping strategies when compared to the previous cohort. Approximately half of the student cohort reported no use of university resources. The most commonly used resources were financial aid and mental health services.Perceived stress decreased following the revision of a Doctor of Pharmacy curriculum. The most common coping strategies were positive and comparable with strategies reported by students in the former curriculum. The impact of curricular changes on student stress and the use of university resources in health professions students warrant further study.

Objective. To describe the development and assessment of an integrated virtual escape room in a cardiology course. Methods. A virtual escape room was developed to reinforce therapeutics, pharmacology, pharmacokinetics, medicinal chemistry, pharmacogenomics, and calculations related to cardiology in an integrated pharmacy course and was completed by two student cohorts. Groups of four to five students had 40 minutes to complete virtual escape room puzzles, and each puzzle had to be solved correctly prior to advancing. After completion of the activity, learners met with facilitators to debrief. Students completed pre- and postsurveys to assess knowledge changes and their perceptions of the experience. Results. One hundred twenty-six second-year Doctor of Pharmacy (PharmD) students completed the escape room, and 79% (n=55) and 93% (n=52) of students completed pre- and postsurveys for the 2020 and 2021 cohorts, respectively. Results showed a significant improvement in student knowledge on pre- to postsurvey knowledge questions (2020 presurvey mean [SD]=43.1 [22.6], postsurvey mean [SD]=74.1 [19.6]; 2021 presurvey mean [SD]=52.0 [15.8], postsurvey mean [SD]=67.1 [19.2]). Most students in both cohorts (88%) agreed that logistics of the escape rooms were amenable to learning and applying information, and 86% enjoyed working through puzzles. Conclusion. The virtual escape room was well received by students and served as an effective tool for reinforcing and integrating cardiology concepts. The virtual nature of the activity makes it practical and easily replicable to implement at other institutions, which can benefit from using the format, logistics, and materials described in this study to decrease faculty workload and costs associated with implementing this educational technique.

Supplementary Figures S1-S2 from Evaluation of Lapatinib and Topotecan Combination Therapy: Tissue Culture, Murine Xenograft, and Phase I Clinical Trial Data

Purpose: Topotecan resistance can result from drug efflux by P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) as well as survival signals initiated by epidermal growth factor receptor family members. The present studies were done to determine the effect of combining topotecan and the dual epidermal growth factor receptor/HER2 inhibitor lapatinib in tissue culture, a murine xenograft model, and a phase I clinical trial.Experimental Design: The effects of lapatinib on topotecan accumulation and cytotoxicity in vitro were examined in paired cell lines lacking or expressing Pgp or BCRP. Antiproliferative effects of the combination were assessed in mice bearing HER2+ BT474 breast cancer xenografts. Based on tolerability in this preclinical model, 37 patients with advanced-stage cancers received escalating doses of lapatinib and topotecan in a phase I trial.Results: Lapatinib increased topotecan accumulation in BCRP- or Pgp-expressing cells in vitro, and the combination showed enhanced efficacy in HER2+ BT474 xenografts. In the phase I study, nausea, vomiting, diarrhea, and fatigue were dose limiting. The maximum tolerated doses were 1,250 mg/d lapatinib by mouth for 21 or 28 days with 3.2 mg/m2 topotecan i.v. on days 1, 8, and 15 of 28-day cycles. Pharmacokinetic analyses showed that combined drug administration resulted in decreased topotecan clearance consistent with transporter-mediated interactions. Seventeen (46%) patients had disease stabilization.Conclusions: The lapatinib/topotecan combination is well tolerated and warrants further study.

d-Pinitol (3-O-methyl-d-chiro-inositol) is a cyclitol nearly ubiquitous in the Leguminosae and Pinaceae families. It plays an important role in plants as physiological cellular modulator and chemical defense against unfavorable environmental conditions, such as water deficit and high level of salinity. Plants rich in d-pinitol are being used in traditional medicine as empirical treatment for diabetes, inflammation, cancer or infections. This review summarizes the extensive literature published about d-pinitol biosynthesis, plant distribution, biological activities and pharmacological studies, including in vitro, in vivo, and clinical trials.

// Joseph R. McDermott 1 , Xiangrong Geng 1 , Lan Jiang 1 , Marina Galvez-Peralta 2 , Fei Chen 3 , Daniel W. Nebert 2 , Zijuan Liu 1 1 Department of Biological Sciences, Oakland University, Rochester, MI 48309, USA 2 Department of Environmental Health and Center for Environmental Genetics, University of Cincinnati Medical Center, Cincinnati, OH 45267, USA 3 Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI 48201, USA Correspondence to: Daniel W. Nebert, email: dan.nebert@uc.edu Zijuan Liu, email: liu2345@oakland.edu Keywords: selenite, zinc, micronutrient, ZIP8, membrane-bound transporter Received: December 26, 2015 Accepted: April 11, 2016 Published: May 6, 2016 ABSTRACT Selenite (HSeO 3 − ) is a monovalent anion of the essential trace element and micronutrient selenium (Se). In therapeutic concentrations, HSeO 3 − has been studied for treating certain cancers, serious inflammatory disorders, and septic shock. Little is known, however, about HSeO 3 − uptake into mammalian cells; until now, no mammalian HSeO 3 − uptake transporter has been identified. The ubiquitous mammalian ZIP8 divalent cation transporter (encoded by the SLC39A8 gene) is bicarbonate-dependent, moving endogenous substrates (Zn 2+ , Mn 2+ , Fe 2+ or Co 2+ ) and nonessential metals such as Cd 2+ into the cell. Herein we studied HSeO 3 − uptake in: human and mouse cell cultures, shRNA-knockdown experiments, Xenopus oocytes, wild-type mice and two transgenic mouse lines having genetically altered ZIP8 expression, and mouse erythrocytes ex vivo . In mammalian cell culture, excess Zn 2+ levels and/or ZIP8 over-expression can be associated with diminished viability in selenite-treated cells. Intraperitoneal HSeO 3 − causes the largest ZIP8-dependent increases in intracellular Se content in liver, followed by kidney, heart, lung and spleen. In every model system studied, HSeO 3 − uptake is tightly associated with ZIP8 protein levels and sufficient Zn 2+ and HCO 3 − concentrations, suggesting that the ZIP8-mediated electroneutral complex transported contains three ions: Zn 2+ /(HCO 3 − )(HSeO 3 − ). Transporters having three different ions in their transport complex are not without precedent. Although there might be other HSeO 3 − influx transporters as yet undiscovered, data herein suggest that mammalian ZIP8 plays a major role in HSeO 3 − uptake.

Slc39a8 encodes ZIP8, a divalent cation/bicarbonate symporter expressed in pluripotent mouse embryonic stem cells, and therefore ubiquitous in adult tissues; ZIP8 influxes Zn2+, Mn2+ and Fe2+. Slc39a8(neo/neo) knockdown mice exhibit 10–15% of wild-type ZIP8 mRNA and protein levels, and show pleiotropic phenotype of stunted growth, neonatal lethality, multi-organ dysmorphogenesis, and dysregulated hematopoiesis manifested as severe anemia. Herein we performed RNA-seq analysis of gestational day (GD)13.5 yolk sac and placenta, and GD16.5 liver, kidney, lung, heart and cerebellum, comparing Slc39a8(neo/neo) with Slc39a8(+/+) wild-type. Meta-data analysis of differentially-expressed genes revealed 29 unique genes from all tissues — having enriched GO categories associated with hematopoiesis and hypoxia and KEGG categories of complement, response to infection, and coagulation cascade — consistent with dysregulated hematopoietic stem cell fate. Based on transcription factor (TF) profiles in the JASPAR database, and searching for TF-binding sites enriched by Pscan, we identified numerous genes encoding zinc-finger and other TFs associated with hematopoietic stem cell functions. We conclude that, in this mouse model, deficient ZIP8-mediated divalent cation transport affects zinc-finger (e.g. GATA proteins) and other TFs interacting with GATA proteins (e.g. TAL1), predominantly in yolk sac. These data strongly support the phenotype of dysmorphogenesis and anemia seen in Slc39a8(neo/neo) mice in utero.

Perceptions and challenges connecting Pharmacogenomics taught in classrooms and translationing it to advance pharmacy practice rotations and healthcare settings and potential areas of development.

The nonessential metal cadmium (Cd) is toxic only after entering the cell. Proteins possibly relevant to intracellular Cd accumulation include the divalent metal transporter-1 (DMT1) and all 14 zinc-like iron-like protein (ZIP) importers, 10 zinc transporter (ZnT) exporters, and metallothionein chaperones MT1 and MT2. Comparing oral Cd-treated ZIP14 knockout (KO) with wild-type (WT) mice, we predicted Cd uptake and distribution would be diminished in the KO—because ZIP14 is very highly expressed in GI tract and liver; this was indeed observed for Cd content in liver. However, the reverse was found in kidney and lung from 6 or 12 h through 10 days of Cd exposure; at these times, Cd accumulation was unexpectedly greater in KO than WT mice; mRNA levels of the 27 above-mentioned genes were thus examined in proximal small intestine (PSI) versus kidney to see if these paradoxical effects could be explained by substantial alterations in any of the other 26 genes. PSI genes highly expressed in untreated WT animals included seven ZIP and five ZnT transporters, DMT1, MT1, and MT2; kidney genes included 11 ZIP and 7 ZnT transporters, DMT1, MT1, and MT2. Over 10 days of oral Cd, a bimodal response was seen for Cd content in PSI and for various mRNAs; initially, acute effects caused by the toxic metal; subsequently, the up- or down-regulation of important genes presumably to combat the sustained adversity. These data underscore the complex interplay between the gastrointestinal tract and renal proteins that might be relevant to Cd uptake and distribution in animals exposed to oral Cd.

Mouse Slc39a8 and Slc39a14 genes encode ZIP8 and ZIP14, respectively, which are ubiquitous divalent cation/(HCO3–)2 symporters responsible for uptake of Zn2+, Fe2+, and Mn2+ into cells. Cd2+ and other toxic nonessential metals can displace essential cations, thereby entering vertebrate cells. Whereas Slc39a8 encodes a single protein, Slc39a14 has 2 exons 4 which, via alternative splicing, give rise to ZIP14A and ZIP14B; why differences exist in cell type-specific expression of ZIP14A and ZIP14B remains unknown. Inflammatory stimuli have been associated with upregulation of ZIP8 and ZIP14, but a systematic study of many tissues simultaneously in a laboratory animal following inflammatory cytokine exposure has not yet been reported. Herein, we show that C57BL/6J male mice––treated intraperitoneally with lipopolysaccharide or the proinflammatory cytokines tumor necrosis factor (TNF) or interleukin-6 (IL6)––exhibited quantatively very different, highly tissue-specific, and markedly time-dependent up- and downregulation of ZIP8, ZIP14A, and ZIP14B messenger RNA (mRNA) levels in 12 tissues. The magnitude of inflammatory response was confirmed by measuring the proinflammatory cytokine TNF, IL6, and interleukin-1β mRNA levels in the same tissues of these animals. Our data suggest that most if not all tissues use ZIP8, ZIP14A, and/or ZIP14B for Zn2+ uptake, some tissues under basal conditions and others moreso when inflammatory stressors are present; collectively, this might lead to substantial alterations in plasma Zn2+ levels due to Zn2+ redistribution not just in liver but across many vital organs. In the context of cadmium-mediated toxicity, our data suggest that tissues other than liver, kidney, and lung should also be considered.

Objective. To develop an effective method in teaching pharmacogenomics as a part of a new course, Biopharmaceutics and Pharmacogenomics. Methods. Teaching effectiveness was measured by quizzes, retrospective pre- and post-surveys, team activities, and journal reflections. Four team activities were included in the course: genomic disease, patient case, genetic counselor and a debate about personalized medicine. Outcomes and course impact were evaluated at the end of the course. The evaluation methods included the assessment of knowledge, students' perceptions regarding the utility of team activities, the impact of the course on students' confidence to discuss pharmacogenomics with health care providers or patients, and long-term knowledge retention, measured in the following P2 semester. Results. Seventy-six students were enrolled in the course. Multiple assessments during the course demonstrated that students' knowledge of pharmacogenomics improved. The team activities had a positive impact on student learning, and the course improved their confidence level to discuss pharmacogenomics with another health care provider or a patient. While 86% of the students considered themselves "unconfident," "somewhat unconfident" or "neither confident nor unconfident" at the beginning of the course, 91% reported being "confident" or "somewhat confident" by the end of the course. This increase in confidence was statistically significant. Furthermore, students showed knowledge retention six months after taking the course. Conclusion. Implementation of a new course in pharmacogenomics was effective and well received by the students. It also prepared students for system-based therapeutics courses later in the curriculum.

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed environmental toxicants derived from sources that include cigarette smoke, petroleum distillation, gas- and diesel-engine exhaust, and charcoal-grilled food. The gastrointestinal tract is the principal route of PAH exposures, even when inhaled. The most thoroughly studied prototype of PAHs is benzo[a]pyrene (BaP), well known to be toxic, mutagenic, and carcinogenic in various tissues and cell types. This lab has previously shown that Cyp1a1(-/-) global knockout mice treated by oral administration of BaP die at 28 to 32 days with immunosuppression, whereas wild-type mice remain healthy for 1 year on high BaP doses (125 mg/kg/day). Thus, for oral BaP, CYP1A1 is more important in detoxication than in metabolic activation. After several days of oral BaP, we found surprisingly low CYP1A1 levels in liver, compared with that in small intestine; we postulated that this finding might reflect efficient detoxication of oral BaP in proximal small intestine such that significant amounts of the inducer BaP no longer reach the liver. In the present study, many parameters were therefore compared in wild-type, Cyp1a1(-/-) global knockout, intestinal epithelial cell-specific Cyp1a1 knockout, and hepatocyte-specific Cyp1a1 knockout mice as a function of long-term oral exposure to BaP. The peak of CYP1A1 (mRNA, protein) expression in liver occurred at 12 h, whereas highly induced CYP1A1 in small intestine persisted throughout the 30-day experiment. Hepatocyte-specific Cyp1a1 knockout mice remained as healthy as wild-type mice; intestinal epithelial cell-specific Cyp1a1 knockout mice behaved like Cyp1a1(-/-) mice, dying with immunosuppression approximately 30 days on oral BaP. We conclude that small intestine CYP1A1, and not liver CYP1A1, is critically important in oral BaP detoxication.

The cytotoxicity and mechanism of action of a series of substituted 9-aminoacridines is evaluated using topoisomerase I and cancer cell growth inhibition assays. In previous work, compounds of this type were shown to catalytically inhibit topoisomerase II, leading to a G1-S phase arrest of the cell cycle and apoptosis in pancreatic cancer cells in vitro and in vivo. The present study expands the potential utility of these compounds in the development of cancer therapeutics by showing that these compounds inhibit proliferation of cell lines derived from the nine most common human cancers. Further results show that at least one of the compounds effectively stabilizes topoisomerase I–DNA adduct formation in intact cells. RNA interference experiments, however, indicate that this interaction does not contribute to the drug-induced killing of cancer cells indicating the compounds may be non-lethal poisons of topoisomerase I.

Purpose: Topotecan resistance can result from drug efflux by P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) as well as survival signals initiated by epidermal growth factor receptor family members. The present studies were done to determine the effect of combining topotecan and the dual epidermal growth factor receptor/HER2 inhibitor lapatinib in tissue culture, a murine xenograft model, and a phase I clinical trial. Experimental Design: The effects of lapatinib on topotecan accumulation and cytotoxicity in vitro were examined in paired cell lines lacking or expressing Pgp or BCRP. Antiproliferative effects of the combination were assessed in mice bearing HER2+ BT474 breast cancer xenografts. Based on tolerability in this preclinical model, 37 patients with advanced-stage cancers received escalating doses of lapatinib and topotecan in a phase I trial. Results: Lapatinib increased topotecan accumulation in BCRP- or Pgp-expressing cells in vitro, and the combination showed enhanced efficacy in HER2+ BT474 xenografts. In the phase I study, nausea, vomiting, diarrhea, and fatigue were dose limiting. The maximum tolerated doses were 1,250 mg/d lapatinib by mouth for 21 or 28 days with 3.2 mg/m2 topotecan i.v. on days 1, 8, and 15 of 28-day cycles. Pharmacokinetic analyses showed that combined drug administration resulted in decreased topotecan clearance consistent with transporter-mediated interactions. Seventeen (46%) patients had disease stabilization. Conclusions: The lapatinib/topotecan combination is well tolerated and warrants further study.

Human DNA topoisomerase I (topo I) is an essential mammalian enzyme that regulates DNA supercoiling during transcription and replication. In addition, topo I is specifically targeted by the anticancer compound camptothecin and its derivatives. Previous studies have indicated that topo I is a phosphoprotein and that phosphorylation stimulates its DNA relaxation activity. The locations of most topo I phosphorylation sites have not been identified, preventing a more detailed examination of this modification. To address this issue, mass spectrometry was used to identify four topo I residues that are phosphorylated in intact cells: Ser10, Ser21, Ser112, and Ser394. Immunoblotting using anti-phosphoepitope antibodies demonstrated that these sites are phosphorylated during mitosis. In vitro kinase assays demonstrated that Ser10 can be phosphorylated by casein kinase II, Ser21 can be phosphorylated by protein kinase Cα, and Ser112 and Ser394 can be phosphorylated by Cdk1. When wild type topo I was pulled down from mitotic cells and dephosphorylated with alkaline phosphatase, topo I activity decreased 2-fold. Likewise, topo I polypeptide with all four phosphorylation sites mutated to alanine exhibited 2-fold lower DNA relaxation activity than wild type topo I after isolation from mitotic cells. Further mutational analysis demonstrated that Ser21 phosphorylation was responsible for this change. Consistent with these results, wild type topo I (but not S21A topo I) exhibited increased sensitivity to camptothecin-induced trapping on DNA during mitosis. Collectively these results indicate that topo I is phosphorylated during mitosis at multiple sites, one of which enhances DNA relaxation activity in vitro and interaction with DNA in cells.

Although agents that inhibit DNA synthesis are widely used in the treatment of cancer, the optimal method for combining such agents and the mechanism of their synergy is poorly understood. The present study examined the effects of combining gemcitabine (2',2'-difluoro 2'-deoxycytidine) and 7-ethyl-10-hydroxycamptothecin (SN-38; the active metabolite of irinotecan), two S-phaseselective agents that individually have broad antitumor activity, in human cancer cells in vitro. Colony-forming assays revealed that simultaneous treatment of Ovcar-5 ovarian cancer cells or BxPC-3 pancreatic cancer cells with gemcitabine and SN-38 resulted in antagonistic effects. In contrast, sequential treatment with these two agents in either order resulted in synergistic anti-proliferative effects, although the mechanism of synergy varied with the sequence. In particular, SN-38 arrested cells in S phase, enhanced the accumulation of gemcitabine metabolites, and diminished checkpoint kinase 1, thereby sensitizing cells in the SN-38 --> gemcitabine sequence. Gemcitabine treatment followed by removal allowed prolonged progression through S phase, contributing to synergy of the gemcitabine --> SN-38 sequence. These results collectively suggest that S-phase-selective agents might exhibit more cytotoxicity when administered sequentially rather than simultaneously.

Signaling through the phosphatidylinositol-3 kinase (PI3K)/Akt pathway, which is aberrantly activated in >50% of carcinomas, inhibits apoptosis and contributes to drug resistance. Accordingly, several Akt inhibitors are currently undergoing preclinical or early clinical testing. To examine the effect of Akt inhibition on the activity of multiple widely used classes of antineoplastic agents, human cancer cell lines were treated with the Akt inhibitor A-443654 [(2S)-1-(1H-indol-3-yl)-3-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxypropan-2-amine; ATP-competitive] or MK-2206 (8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-2H-[1,2,4]triazolo[3,4-f][1,6]naphthyridin-3-one;dihydrochloride; allosteric inhibitor) or with small interfering RNA (siRNA) targeting phosphoinositide-dependent kinase 1 (PDK1) along with cisplatin, melphalan, camptothecin, or etoposide and assayed for colony formation. Surprisingly different results were observed when Akt inhibitors were combined with different drugs. Synergistic effects were observed in multiple cell lines independent of PI3K pathway status when A-443654 or MK-2206 was combined with the DNA cross-linking agents cisplatin or melphalan. In contrast, effects of the Akt inhibitors in combination with camptothecin or etoposide were more complicated. In HCT116 and DLD1 cells, which harbor activating PI3KCA mutations, A-443654 over a broad concentration range enhanced the effects of camptothecin or etoposide. In contrast, in cell lines lacking activating PI3KCA mutations, partial inhibition of Akt signaling synergized with camptothecin or etoposide, but higher A-443654 or MK-2206 concentrations (>80% inhibition of Akt signaling) or PDK1 siRNA antagonized the topoisomerase poisons by diminishing DNA synthesis, a process that contributes to effective DNA damage and killing by these agents. These results indicate that the effects of combining inhibitors of the PI3K/Akt pathway with certain classes of chemotherapeutic agents might be more complicated than previously recognized.

All three cytochrome P450 1 (CYP1) monooxygenases are believed to participate in lipid mediator biosynthesis and/or their local inactivation; however, distinct metabolic steps are unknown. We used multiple-reaction monitoring and liquid chromatography-UV coupled with tandem mass spectrometry–based lipid-mediator metabololipidomics to identify and quantify three lipid-mediator metabolomes in basal peritoneal and zymosan-stimulated inflammatory exudates, comparing Cyp1a1/1a2/1b1(−/−) C57BL/6J-background triple-knockout mice with C57BL/6J wild-type mice. Significant differences between untreated triple-knockout and wild-type mice were not found for peritoneal cell number or type or for basal CYP1 activities involving 11 identified metabolic steps. Following zymosan-initiated inflammation, 18 lipid mediators were identified, including members of the eicosanoids and specialized proresolving mediators (i.e., resolvins and protectins). Compared with wild-type mice, Cyp1 triple-knockout mice exhibited increased neutrophil recruitment in zymosan-treated peritoneal exudates. Zymosan stimulation was associated with eight statistically significantly altered metabolic steps: increased arachidonic acid–derived leukotriene B4 (LTB4) and decreased 5S-hydroxyeicosatetraenoic acid; decreased docosahexaenoic acid–derived neuroprotectin D1/protectin D1, 17S-hydroxydocosahexaenoic acid, and 14S-hydroxydocosahexaenoic acid; and decreased eicosapentaenoic acid–derived 18R-hydroxyeicosapentaenoic acid (HEPE), 15S-HEPE, and 12S-HEPE. In neutrophils analyzed ex vivo, elevated LTB4 levels were shown to parallel increased neutrophil numbers, and 20-hydroxy–LTB4 formation was found to be deficient in Cyp1 triple-knockout mice. Together, these results demonstrate novel contributions of CYP1 enzymes to the local metabolite profile of lipid mediators that regulate neutrophilic inflammation.

Benzo[a]pyrene (BaP) is a prototypical polycyclic aromatic hydrocarbon (PAH); this ubiquitous environmental carcinogenic agent is found in tobacco smoke, charcoal-grilled foods, and PAH-contaminated surfaces of roofs, playgrounds, and highways. Cytochrome P450 1 wild-type, Cyp1a2(−/−), Cyp1b1(−/−), or Cyp1a2/1b1(−/−) knockouts, and mice with Cyp1a1 expression deleted in hepatocytes can ingest large oral BaP doses (125 mg/kg/d) without apparent toxicity. Cyp1a1(−/−) and Cyp1a1/1a2(−/−) knockouts and mice with Cyp1a1 expression deleted in gastrointestinal (GI) tract epithelial cells develop immunotoxicity and die within 32 days, indicating that GI tract inducible CYP1A1 is absolutely required for detoxication of oral BaP. Cyp1a1/1b1(−/−) and Cyp1a1/1a2/1b1(−/−) mice are rescued from immunosuppression and early death due to absent metabolic activation of BaP by CYP1B1 in immune cells. Ten-fold lower oral BaP doses result in adenocarcinoma of the proximal small intestine (PSI) in Cyp1a1(−/−) mice; Cyp1a1/1b1(−/−) double-knockout mice show no PSI cancer but develop squamous cell carcinoma of the preputial gland duct (PGD). BaP-metabolizing CYP1B1 in the PSI and CYP3A59 in the PGD are the most likely candidates to participate in tumor initiation in the epithelial cells of these two tissues; oncogenes and tumor-suppressor genes upregulated and downregulated during tumorigenesis are completely different between these tissues. This “oral BaP Cyp1” mouse paradigm represents a powerful teaching tool, showing that gene-environment interactions depend on route-of-administration: the same oral, but not intraperitoneal, BaP exposure leads to dramatic differences in target-organ toxicity and tumor type as a function of dose and Cyp1 genotype.

Benzo[a]pyrene (BaP) is a prototypical polycyclic aromatic hydrocarbon (PAH) found in combustion processes. Cytochrome P450 1A1 and 1B1 enzymes (CYP1A1, CYP1B1) and other enzymes can activate PAHs to reactive oxygenated intermediates involved in mutagenesis and tumor initiation; also, CYP1 enzymes can detoxify PAHs. Cyp1(+/+) wild-type (WT) and Cyp1b1(-/-) knockout mice receiving oral BaP (12.5 mg/kg/day) remain healthy for >12 months. In contrast, we found that global knockout of the Cyp1a1 gene (1a1KO) results in proximal small intestine (PSI) adenocarcinoma within 8-12 weeks on this BaP regimen; striking compensatory increases in PSI CYP1B1 likely participate in initiation of adenocarcinoma in 1a1KO mice. Cyp1a1/1b1(-/-) double-knockout (DKO) mice on this BaP regimen show no PSI adenocarcinoma, but instead preputial gland duct (PGD) squamous cell carcinoma (SCC) occurs by 12 weeks. Herein, we compare microarray expression of PGD genes in WT, 1a1KO and DKO mice at 0, 4, 8, 12 and 16 weeks of oral BaP; about four dozen genes up- or down-regulated during most critical time-points were further verified by qRT-PCR. In DKO mice, CYP3A59 was unequivocally identified as the BaP-inducible and BaP-metabolizing best candidate responsible for initiation of BaP-induced SCC. Striking increases or decreases were found in 26 cancer-related genes plus eight Serpin genes in DKO, but not in 1a1KO or WT, mice on this BaP regimen; of the 26, 8 were RAS-related oncogenes. The mechanism by which cancer-related genes are responsible for SCC tumor progression in the PGD remains to be elucidated.

Previously this laboratory characterized Slc39a8-encoded ZIP8 as a Zn(2+)/(HCO(3)(-))(2) symporter; yet, the overall physiological importance of ZIP8 at the whole-organism level remains unclear. Herein we describe the phenotype of the hypomorphic Slc39a8(neo/neo) mouse which has retained the neomycin-resistance gene in intron 3, hence causing significantly decreased ZIP8 mRNA and protein levels in embryo, fetus, placenta, yolk sac, and several tissues of neonates. The Slc39a8(neo) allele is associated with diminished zinc and iron uptake in mouse fetal fibroblast and liver-derived cultures; consequently, Slc39a8(neo/neo) newborns exhibit diminished zinc and iron levels in several tissues. Slc39a8(neo/neo) homozygotes from gestational day(GD)-11.5 onward are pale, growth-stunted, and die between GD18.5 and 48 h postnatally. Defects include: severely hypoplastic spleen; hypoplasia of liver, kidney, lung, and lower limbs. 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 severe anemia. Flow cytometry of fetal liver cells revealed the erythroid series strikingly affected in the hypomorph. Zinc-dependent 5-aminolevulinic acid dehydratase, required for heme synthesis, was not different between Slc39a8(+/+) and Slc39a8(neo/neo) offspring. To demonstrate further that the mouse phenotype is due to ZIP8 deficiency, we bred Slc39a8(+/neo) with BAC-transgenic BTZIP8-3 line (carrying three extra copies of the Slc39a8 allele); this cross generated viable Slc39a8(neo/neo)_BTZIP8-3(+/+) pups showing none of the above-mentioned congenital defects-proving Slc39a8(neo/neo) causes the described phenotype. Our study demonstrates that ZIP8-mediated zinc transport plays an unappreciated critical role during in utero and neonatal growth, organ morphogenesis, and hematopoiesis.

The highly conserved human and mouse SLC39A14 and SLC39A8 genes encode the ZIP14 and ZIP8 transporters, respectively-functioning as divalent cation/bicarbonate symporters and expressed in dozens of tissues. Due to alternative splicing of exons 4, human and mouse SLC39A14 genes each encode two distinct gene products, whereas SLC39A8 produces a single product. This lab previously noted that ZIP14A and ZIP14B show highly variable expression in different cell types, suggesting differences in metal uptake function. We ligated mouse ZIP14A, ZIP14B and ZIP8 cDNA coding regions into the Xenopus-specific vector pXFRM, transcribed these in vitro, and microinjected the capped RNAs into Xenopus oocytes. K(m) and V(max) values for Cd, Zn and Fe uptake were determined. Electrogenicity studies using a potassium gradient confirmed that (just as we found previously for ZIP8) ZIP14A- and ZIP14B-mediated divalent Cd- or Zn-bicarbonate complexes are electroneutral. Competitive inhibition of Cd and Zn uptake with ten additional divalent cations showed a unique gradient of patterns for each of ZIP14A, ZIP14B and ZIP8. ZIP14 proteins are prominent in the gastrointestinal tract and ZIP8 protein is located on the surface of renal proximal tubular epithelial cells. It is known that renal Fanconi syndrome can be caused by five nonessential heavy metals: Cd(2+), Hg(2+), Pb(2+), Pt(2+) and U(2+). In the present study we show that these five divalent cations are usually competitors of ZIP14- and/or ZIP8-mediated Zn uptake; our data thus support the possible involvement of intestinal ZIP14 for uptake of these five metals into the body and ZIP8 for efficient uptake into the kidney.

Zinc is an essential micronutrient required for proper immune function. We previously reported that zinc deficiency increases inflammation and mortality in response to polymicrobial sepsis. Here we report that a specific zinc transporter, SLC39A8 (ZIP8), is a transcriptional target of NF-κB and functions as a negative regulator of NF-κB. Initially we observed that TNFα or LPS inducesSLC39A8 expression in human monocytes, macrophages and lung epithelial cells, leading to an increase in intracellular zinc. We then identified a functional NF-κB binding site in the promoter region that triggers ZIP8 expression. Silencing of ZIP8 expression significantly decreased intracellular accumulation of zinc and increased the inflammatory response, as determined by increased cytokine production and phosphorylation of IκBα, P65, ERK and Akt. We further revealed that zinc directly inhibits IKKβ in a ZIP8-dependent manner. In mice, endotoxin or cecal ligation and puncture resulted in increased ZIP8 expression and zinc uptake in circulating monocytes and lung tissue, contributing to hypozincemia. Consistent with these observations, mouse embryonic fibroblasts obtained from Slc39a8 hypomorphic mice were more responsive to TNFα and IL-1β compared to wild-type cells, confirming the function of ZIP8 as a negative regulator of NF-κB and innate immune activation. Taken together, our findings identify a novel inducible inhibitory feedback pathway for NF-κB and the pro-inflammatory response.

The correspondence article by Christian Biemont (Biemont, C. Inbreeding effects in the epigenetic era. Nature Rev. Genet. 11, 234 (2010))1 and the articles (Charlesworth, D. & Willis J. H. The genetics of inbreeding depression. Nature Rev. Genet. 10, 783–796 (2009)2,3 he discusses deserve further dialogue. These papers summarized the theoretical basis of inbreeding depression, defined as the deleterious effects that result from crossing related individuals. Rather than this phenomenon being the result of DNA sequence alterations, Biemont puts forth a strong argument1 that inbreeding depression probably results from epigenetic mechanisms — that is, chromosomal events in which DNA sequence is not altered. Epigenetic mechanisms include cell-specific DNA hyper- and hypomethylation4, RNA interference5, histone modifications6, chromatin remodelling7 and canalization/decanalization8. Our laboratory has observed the opposite of inbreeding depression — that is, ‘inbreeding de-repression’. This can be defined as the beneficial effects that result from crossing related individuals. By inbreeding a freshly generated knockout mouse line, we found that subsequent generations produced much healthier viable offspring compared with the F1 generation. Cytochrome P450 genes, or at least those members in the Cyp1, Cyp2, Cyp3 and Cyp4 gene families9, are commonly perceived as liver enzymes that metabolize drugs and environmental chemicals. However, most if not all of the CYP1 to CYP4 enzymes exist in many cell types and have important endogenous roles. Among other functions, these enzymes participate in eicosanoid synthesis and/or degradation9,10 and therefore are probably crucial upstream determinants of innumerable cellular processes, from vascular or airway constriction/relaxation to tumour progression and cancer metastasis10. Our laboratory generated the Cyp1a1–Cyp1a2–Cyp1b1–/– triple-knockout mouse by crossing various combinations of female versus male Cyp1a1–Cyp1a2+/– double-knockout heterozygotes with Cyp1b1+/– single-knockout heterozygotes. From 150 F1 litters examined, 58 Cyp1a1–Cyp1a2–Cyp1b1–/– pups survived to birth, and only two females and one male lived to adulthood and were able to breed successfully11. Incomplete-penetrance phenotypes seen in the F1 generation included embryolethality, slower weight gain, hydrocephalus, hermaphroditism and cystic ovaries11. All of these phenotypes are consistent with eicosanoid-mediated processes10,11. Most intriguingly, as we continued to breed the triple-knockout F1 homozygote survivors, the embryolethality, the lower weight and all of the above-mentioned birth defects disappeared quickly. We believe that these findings represent the action of natural selection — that is, only the healthiest animals survived and were able to breed the subsequent generation. Because this inbreeding de-repression phenomenon happened within just two to four generations, we presume it reflects epigenetic rather than genetic changes in the genome. Although we suspect this also has occurred with other knockout mouse lines, we are not aware of this ever having been reported. In conclusion, inbreeding can lead not only to depression but also to an improved, healthier, viable phenotype. And, in all likelihood, both deleterious and beneficial traits that appear during brother–sister inbreeding are caused by epigenetic rather than genetic mechanisms.

SummaryActivation of the transcription factor NF-κB is essential for innate immune function and requires strict regulation. The micronutrient zinc modulates proper host defense, and zinc deficiency is associated with elevated inflammation and worse outcomes in response to bacterial infection and sepsis. Previous studies suggest that zinc may regulate NF-κB activity during innate immune activation, but a mechanistic basis to support this has been lacking. Herein, we report that the zinc transporter SLC39A8 (ZIP8) is a transcriptional target of NF-κB and functions to negatively regulate proinflammatory responses through zinc-mediated down-modulation of IκB kinase (IKK) activity in vitro. Accordingly, fetal fibroblasts obtained from Slc39a8 hypomorphic mice exhibited dysregulated zinc uptake and increased NF-κB activation. Consistent with this, mice provided zinc-deficient dietary intakes developed excessive inflammation to polymicrobial sepsis in conjunction with insufficient control of IKK. Our findings identify a negative feedback loop that directly regulates innate immune function through coordination of zinc metabolism.