Impact of phenanthrene co-administration on the toxicokinetics of benzo[a]pyrene in humans. UPLC-accelerator mass spectrometry following oral microdosing.

Current risk assessments for environmental carcinogens rely on animal studies utilizing doses orders of magnitude higher than actual human exposures. Epidemiological studies of people with high exposures (e.g., occupational) are of value, but rely on uncertain exposure data. In addition, exposures are typically not to a single chemical but to mixtures, such as polycyclic aromatic hydrocarbons (PAHs). The extremely high sensitivity of accelerator mass spectrometry (AMS) allows for dosing humans with known carcinogens with de minimus risk. In this study UPLC-AMS was used to assess the toxicokinetics of [14C]-benzo[a]pyrene ([14C]-BaP) when dosed alone or in a binary mixture with phenanthrene (Phe). Plasma was collected for 48 h following a dose of [14C]-BaP (50 ng, 5.4 nCi) or the same dose of [14C]-BaP plus Phe (1250 ng). Following the binary mixture, Cmax of [14C]-BaP significantly decreased (4.4-fold) whereas the volume of distribution (Vd) increased (2-fold). Further, the toxicokinetics of twelve [14C]-BaP metabolites provided evidence of little change in the metabolite profile of [14C]-BaP and the pattern was overall reduction consistent with reduced absorption (decrease in Cmax). Although Phe was shown to be a competitive inhibitor of the major hepatic cytochrome P-450 (CYP) responsible for metabolism of [14C]-BaP, CYP1A2, the high inhibition constant (Ki) and lack of any increase in unmetabolized [14C]-BaP in plasma makes this mechanism unlikely to be responsible. Rather, co-administration of Phe reduces the absorption of [14C]-BaP through a mechanism yet to be determined. This is the first study to provide evidence that, at actual environmental levels of exposure, the toxicokinetics of [14C]-BaP in humans is markedly altered by the presence of a second PAH, Phe, a common component of environmental PAH mixtures.

Copper delivery to the CNS by CuATSM effectively treats motor neuron disease in SOD(G93A) mice co-expressing the Copper-Chaperone-for-SOD.

Over-expression of mutant copper, zinc superoxide dismutase (SOD) in mice induces ALS and has become the most widely used model of neurodegeneration. However, no pharmaceutical agent in 20 years has extended lifespan by more than a few weeks. The Copper-Chaperone-for-SOD (CCS) protein completes the maturation of SOD by inserting copper, but paradoxically human CCS causes mice co-expressing mutant SOD to die within two weeks of birth. Hypothesizing that co-expression of CCS created copper deficiency in spinal cord, we treated these pups with the PET-imaging agent CuATSM, which is known to deliver copper into the CNS within minutes. CuATSM prevented the early mortality of CCSxSOD mice, while markedly increasing Cu, Zn SOD protein in their ventral spinal cord. Remarkably, continued treatment with CuATSM extended the survival of these mice by an average of 18 months. When CuATSM treatment was stopped, these mice developed ALS-related symptoms and died within 3 months. Restoring CuATSM treatment could rescue these mice after they became symptomatic, providing a means to start and stop disease progression. All ALS patients also express human CCS, raising the hope that familial SOD ALS patients could respond to CuATSM treatment similarly to the CCSxSOD mice.

The influences of parental diet and vitamin E intake on the embryonic zebrafish transcriptome.

The composition of the typical commercial diet fed to zebrafish can dramatically vary. By utilizing defined diets we sought to answer two questions: 1) How does the embryonic zebrafish transcriptome change when the parental adults are fed a commercial lab diet compared with a sufficient, defined diet (E+)? 2) Does a vitamin E-deficient parental diet (E-) further change the embryonic transcriptome? We conducted a global gene expression study using embryos from zebrafish fed a commercial (Lab), an E+ or an E- diet. To capture differentially expressed transcripts prior to onset of overt malformations observed in E- embryos at 48h post-fertilization (hpf), embryos were collected from each group at 36hpf. Lab embryos differentially expressed (p<0.01) 946 transcripts compared with the E+ embryos, and 2656 transcripts compared with the E- embryos. The differences in protein, fat and micronutrient intakes in zebrafish fed the Lab compared with the E+ diet demonstrate that despite overt morphologic consistency, significant differences in gene expression occurred. Moreover, functional analysis of the significant transcripts in the E- embryos suggested perturbed energy metabolism, leading to overt malformations and mortality. Thus, these findings demonstrate that parental zebrafish diet has a direct impact on the embryonic transcriptome.

Chronic vitamin E deficiency promotes vitamin C deficiency in zebrafish leading to degenerative myopathy and impaired swimming behavior.

We hypothesized that zebrafish (Danio rerio) undergoing long-term vitamin E deficiency with marginal vitamin C status would develop myopathy resulting in impaired swimming. Zebrafish were fed for 1 y a defined diet without (E-) and with (E+) vitamin E (500 mg α-tocopherol/kg diet). For the last 150 days, dietary ascorbic acid concentrations were decreased from 3500 to 50 mg/kg diet and the fish sampled periodically to assess ascorbic acid concentrations. The ascorbic acid depletion curves were faster in the E- compared with E+ fish (P < 0.0001); the estimated half-life of depletion in the E- fish was 34 days, while in it was 55 days in the E+ fish. To assess swimming behavior, zebrafish were monitored individually following a "startle-response" stimulus, using computer and video technology. Muscle histopathology was assessed using hematoxylin and eosin staining on paramedian sections of fixed zebrafish. At study end, E- fish contained 300-fold less α-tocopherol (p < 0.0001), half the ascorbic acid (p = 0.0001) and 3-fold more malondialdehyde (p = 0.0005) than did E+ fish. During the first minute following a tap stimulus (p < 0.05), E+ fish swam twice as far as did E- fish. In the E- fish, the sluggish behavior was associated with a multifocal, polyphasic, degenerative myopathy of the skeletal muscle. The myopathy severity ranged from scattered acute necrosis to widespread fibrosis and was accompanied by increased anti-hydroxynonenal staining. Thus, vitamin E deficiency in zebrafish causes increased oxidative stress and a secondary depletion of ascorbic acid, resulting in severe damage to muscle tissue and impaired muscle function.

The α-tocopherol transfer protein is essential for vertebrate embryogenesis.

The hepatic α-tocopherol transfer protein (TTP) is required for optimal α-tocopherol bioavailability in humans; mutations in the human TTPA gene result in the heritable disorder ataxia with vitamin E deficiency (AVED, OMIM #277460). TTP is also expressed in mammalian uterine and placental cells and in the human embryonic yolk-sac, underscoring TTP's significance during fetal development. TTP and vitamin E are essential for productive pregnancy in rodents, but their precise physiological role in embryogenesis is unknown. We hypothesize that TTP is required to regulate delivery of α-tocopherol to critical target sites in the developing embryo. We tested to find if TTP is essential for proper vertebrate development, utilizing the zebrafish as a non-placental model. We verify that TTP is expressed in the adult zebrafish and its amino acid sequence is homologous to the human ortholog. We show that embryonic transcription of TTP mRNA increases >7-fold during the first 24 hours following fertilization. In situ hybridization demonstrates that Ttpa transcripts are localized in the developing brain, eyes and tail bud at 1-day post fertilization. Inhibiting TTP expression using oligonucleotide morpholinos results in severe malformations of the head and eyes in nearly all morpholino-injected embryos (88% compared with 5.6% in those injected with control morpholinos or 1.7% in non-injected embryos). We conclude that TTP is essential for early development of the vertebrate central nervous system.

Vitamin E decreases extra-hepatic menaquinone-4 concentrations in rats fed menadione or phylloquinone.

SCOPE: The mechanism for increased bleeding and decreased vitamin K status accompanying vitamin E supplementation is unknown. We hypothesized that elevated hepatic α-tocopherol (α-T) concentrations may stimulate vitamin K metabolism and excretion. Furthermore, α-T may interfere with the side chain removal of phylloquinone (PK) to form menadione (MN) as an intermediate for synthesis of tissue-specific menaquinone-4 (MK-4). METHODS AND RESULTS: In order to investigate these hypotheses, rats were fed phylloquinone (PK) or menadione (MN) containing diets (2 µmol/kg) for 2.5 weeks. From day 10, rats were given daily subcutaneous injections of either α-T (100 mg/kg) or vehicle and were sacrificed 24 h after the seventh injection. Irrespective of diet, α-T injections decreased MK-4 concentrations in brain, lung, kidney, and heart; and PK in lung. These decreases were not accompanied by increased excretion of urinary 5C- or 7C-aglycone vitamin K metabolites, however, the urinary α-T metabolite (α-CEHC) increased ≥ 100-fold. Moreover, α-T increases were accompanied by downregulation of hepatic cytochrome P450 expression and modified expression of tissue ATP-binding cassette transporters. CONCLUSION: Thus, in rats, high tissue α-T depleted tissue MK-4 without significantly increasing urinary vitamin K metabolite excretion. Changes in tissue MK-4 and PK levels may be a result of altered regulation of transporters.

Zebrafish (Danio rerio) fed vitamin E-deficient diets produce embryos with increased morphologic abnormalities and mortality.

Vitamin E (α-tocopherol) is required to prevent fetal resorption in rodents. To study α-tocopherol's role in fetal development, a nonplacental model is required. Therefore, the zebrafish, an established developmental model organism, was studied by feeding the fish a defined diet with or without added α-tocopherol. Zebrafish (age, 4-6 weeks) were fed the deficient (E-), sufficient (E+) or lab diet up to 1 years. All groups showed similar growth rates. The exponential rate of α-tocopherol depletion up to ~80 day in E- zebrafish was 0.029±0.006 nmol/g, equivalent to a depletion half-life of 25±5 days. From age ~80 days, the E- fish (5±3 nmol/g) contained ~50 times less α-tocopherol than the E+ or lab diet fish (369±131 or 362±107, respectively; P<.05). E-depleted adults demonstrated decreased startle response suggesting neurologic deficits. Expression of selected oxidative stress and apoptosis genes from livers isolated from the zebrafish fed the three diets were evaluated by quantitative polymerase chain reaction and were not found to vary with vitamin E status. When E-depleted adults were spawned, they produced viable embryos with depleted α-tocopherol concentrations. The E- embryos exhibited a higher mortality (P<.05) at 24 h post-fertillization and a higher combination of malformations and mortality (P<.05) at 120 h post-fertillization than embryos from parents fed E+ or lab diets. This study documents for the first time that vitamin E is essential for normal zebrafish embryonic development.

Vitamin E deficiency decreases long-chain PUFA in zebrafish (Danio rerio).

α-Tocopherol is a required, lipid-soluble antioxidant that protects PUFA. We hypothesized that α-tocopherol deficiency in zebrafish compromises PUFA status. Zebrafish were fed for 1 y either an α-tocopherol-sufficient (E+; 500 mg α-tocopherol/kg) or -deficient (E-; 1.1 mg α-tocopherol/kg) diet containing α-linolenic (ALA) and linoleic (LA) acids but without arachidonic acid (ARA), EPA, or DHA. Vitamin E deficiency in zebrafish decreased by ~20% (n-6) (P < 0.05) and (n-3) (P < 0.05) PUFA and increased the (n-6):(n-3) PUFA ratio (P < 0.05). In E- compared to E+ females, long chain-PUFA status was impaired, as assessed by a ~60% lower DHA:ALA ratio (P < 0.05) and a ~50% lower ARA:LA ratio (P < 0.05). fads2 (P < 0.05) and elovl2 (P < 0.05) mRNA expression was doubled in E- compared to E+ fish. Thus, inadequate vitamin E status led to a depletion of PUFA that may be a result of either or both increased lipid peroxidation and an impaired ability to synthesize sufficient PUFA, especially (n-3) PUFA.

α-Tocopherol injections in rats up-regulate hepatic ABC transporters, but not cytochrome P450 enzymes.

The role of hepatic xenobiotic regulatory mechanisms in modulating hepatic α-tocopherol concentrations during excess vitamin E administration remains unclear. We hypothesized that increased hepatic α-tocopherol would cause a marked xenobiotic response. Thus, we assessed cytochrome P450 oxidation systems (phase I), conjugation systems (phase II), and transporters (phase III) after daily α-tocopherol injections (100mg/kg body wt) for up to 9days in rats. α-Tocopherol injections increased hepatic α-tocopherol concentrations nearly 20-fold, along with a 10-fold increase in the hepatic α-tocopherol metabolites α-CEHC and α-CMBHC. Expression of phase I (CYP3A2, CYP3A1, CYP2B2) and phase II (SULT2A1) proteins and/or mRNAs was variably affected by α-tocopherol injections; however, expression of phase III transporter genes was consistently changed by α-tocopherol. Two liver efflux transporter genes, ABCB1b and ABCG2, were up-regulated after α-tocopherol injections, whereas OATP, a liver influx transporter, was down-regulated. Thus, an overload of hepatic α-tocopherol increases its own metabolism and increases expression of genes of transporters that are postulated to lead to increased excretion of both vitamin E and its metabolites.

Intestinal overexpression of ZNF148 suppresses ApcMin/+ neoplasia.

ZNF148 (ZBP-89, Zfp148) is a multifunctional transcription factor expressed at low levels in most tissues. When overexpressed in gastrointestinal cancer cell lines, ZNF148 inhibits cellular proliferation and induces apoptosis. We sought to determine whether intestinal ZNF148 overexpression would abrogate adenoma development in the ApcMin/+ mouse, i.e., whether ZNF148 is a tumor suppressor. The 13-kb villin promoter was spliced upstream of the ZNF148 cDNA to generate transgenic villin-ZNF148 (ZNF148TgVZ) mice. Intestinal mucosal ZNF148 expression was elevated in four of five ZNF148(TgVZ) lineages and correlated with increased caspase-3 expression and activation. In addition, DNA fragmentation was increased in ZNF148TgVZ mice relative to wild-type littermates. These results suggested that increased intestinal ZNF148 expression induces apoptosis. ZNF148TgVZ mice were crossed with ApcMin/+ mice to assess the biological significance of intestinal ZNF148 overexpression. The presence of the ZNF148TgVZ allele in ApcMin/+ mice correlated with reduced gastrointestinal bleeding at 5 weeks, a 50% reduction in adenoma burden at 20-22 weeks, and prolonged survival (median survival of 33.5 days vs. 21.5 days), relative to nontransgenic littermates. These data suggest that enhanced ZNF148 expression activates intestinal apoptosis and thereby mitigates disease burden in ApcMin/+ mice. They also suggest that ZNF148 is a therapeutic target to inhibit colon cancer development.

An isoform of ZBP-89 predisposes the colon to colitis.

Alternative splicing enables expression of functionally diverse protein isoforms. The structural and functional complexity of zinc-finger transcription factor ZBP-89 suggests that it may be among the class of alternatively spliced genes. We identified a human ZBP-89 splice isoform (ZBP-89(DeltaN)), which lacks amino terminal residues 1-127 of the full-length protein (ZBP-89(FL)). ZBP-89(DeltaN) mRNA was co-expressed with its ZBP-89(FL) cognate in gastrointestinal cell lines and tissues. Similarly, ZBP-89(DeltaN) protein was expressed. To define its function in vivo, we generated ZBP-89(DeltaN) knock-in mice by targeting exon 4 that encodes the amino terminus. Homozygous ZBP-89(DeltaN) mice, expressing only ZBP-89(DeltaN) protein, experienced growth delay, reduced viability and increased susceptibility to dextran sodium sulfate colitis. We conclude that ZBP-89(DeltaN) antagonizes ZBP-89(FL) function and that over-expression of the truncated isoform disrupts gastrointestinal homeostasis.