Charles C. McCormick

Molecular Cloning and Expression of an Avian Macrophage Nitric-oxide Synthase cDNA and the Analysis of the Genomic 5′-Flanking Region

We report the first nonmammalian inducible nitric-oxide synthase (NOS) cDNA obtained from chicken macrophages. It exhibits an open reading frame encoding 1,136 amino acid residues, predicting a protein of 129,648-Da molecular mass. The deduced NOS protein sequence showed 66.6%, 70.4%, 54.2%, and 48.7% sequence identity to mouse and human inducible NOS and to two constitutive NOSs from rat brain and bovine endothelium. Overall, NOS appears to be a moderately conserved protein. Northern analysis showed that chicken iNOS mRNA is approximately 4.5 kilobases (kb), a size similar to mammalian inducible NOS. Analysis of 3.2 kb of 5′-flanking sequence of the chicken iNOS gene showed a putative TATA box at 30 base pairs (bp) upstream of the transcription initiation site. The functional importance of the upstream region was determined by transient expression of deletion constructs. An endotoxin regulatory region was located exclusively within 300 bp upstream of the transcription initiation site. This is in contrast to the two distinct sites identified in the mouse macrophage NOS promoter. Transcription factor binding sites such as NF-κB, PEA1, PEA3, and C/EBP were identified. Using a NF-κB inhibitor, we showed that NF-κB is indeed involved in the induction of chicken iNOS gene by lipopolysaccharide. Our results suggest that NF-κB is a common regulatory component in the expression of both mammalian and nonmammalian iNOS genes.

Discovery, Characterization, and Significance of the Cytochrome P450 ω‐Hydroxylase Pathway of Vitamin E Catabolism

Abstract: Tocopherols are known to undergo metabolism to phytyl chain‐shortened metabolites excreted in urine. We sought to characterize the pathway, including associated enzymes, involved in this biotransformation. We previously found that human hepatoblastoma (HepG2) cultures metabolized tocopherols to their corresponding short‐chain carboxychromanols. Putative metabolites of γ‐tocopherol that contained intact chromanol moieties were structurally identified using HepG2 cultures and electron impact gas chromatography‐mass spectrometry. A microsomal assay for synthesis of the initial ω‐oxidation metabolites was developed and used to screen several recombinant human liver cytochrome P450 isozymes for ω‐hydroxylase activity. Seven metabolites of γ‐tocopherol were identified in HepG2 cultures, including 13′‐hydroxy‐γ‐TOH and all six carboxychromanols predicted by sequential ω‐oxidation truncation. Rat and human liver microsomes catalyzed synthesis of 13′‐OH‐ and 13′‐COOH‐γ‐TOH, but not other metabolites, in the presence of NADPH. Inclusion of NAD favored synthesis of the 13′‐COOH metabolite. Recombinant CYP4F2, but not other major human liver CYP isoforms (including CYP3A4 and 3A7), exhibited tocopherol‐ω‐hydroxylase activity. Liver microsomes and recombinant CYP4F2 both exhibited substrate preference for γ‐TOH over α‐TOH, and recent studies show that tocotrienols are catabolized more extensively than the corresponding tocopherols. Comparative rates of ω‐oxidation of tocochromanols in hepatocytes are inversely related to biopotency and directly related to cytotoxicity of these substances in macrophages. The liver contains a cytochrome P450‐mediated pathway that preferentially catabolizes “non‐α” tocochromanols to excretable metabolites. This metabolic pathway appears central to the optimization of tissue tocochromanol status.

The Tissue-Specific Accumulation of Hepatic Zinc Metallothionein following Parenteral Iron Loading

Abstract The synthesis in various tissues of the unique metal-binding protein, metallothionein, can be influenced by the administration of certain trace elements. Zinc and cadmium, both of which bind to metallothionein, are most widely recognized as potent inducers. Preliminary results in our laboratory suggested that iron loading causes a marked accumulation of hepatic zinc metallothionein. We present in this report the effects of parenteral iron administration on metallothionein concentration in various tissues. Male chicks (300-350 g) received (ip) either a single injection (+1 Fe) of iron (10 mg Fe/kg, as FeCl3), two injections (+2 Fe) given 24-hr apart, three injections (+3 Fe) each given 24-hr apart, or an equivalent volume of 0.9% saline (control). Twenty-four hours following the final injection, chicks were killed and tissues analyzed for cytoplasmic zinc and metallothionein (Zn-MT). The parenteral administration of ferric iron, FeCl3, resulted in a marked tissue-specific accumulation of zinc as metallothionein. In chicks given +2 Fe, hepatic Zn-MT increased more than 10-fold with a third injection (+3 Fe) causing no further change. The concentration of Zn-MT in renal and pancreatic tissue was unaffected by iron loading. An increase in hepatic Zn-MT was evident prior to detectable changes in total hepatic iron. The administration of other ferrous iron compounds at a similar rate produced comparable changes in hepatic Zn-MT. Feeding excess dietary iron, however, had no effect on liver Zn-MT levels even though similar hepatic iron concentrations were attained. Our results indicated that parenteral administration, but not feeding, of various iron compounds causes a marked increase in zinc metallothionein, specifically in liver tissue.

Lipopolysaccharide and interferon-γ-induced nitric oxide production and protein oxidation in mouse peritoneal macrophages are affected by glutathione peroxidase-1 gene knockout

This study investigated the role of glutathione peroxidase-1 (GPX1) in protein oxidation in peritoneal macrophages. Macrophages isolated from both wild-type (WT) and GPX1 knockout (KO) mice were activated by lipopolysaccharide (LPS, 1 microg/ml) and interferon-gamma (IFN, 10 U/ml for 24 or 48 h in the presence or absence of 1 microM diquat (DQ), 250 microM aminoguanidine (AG, an inhibitor of inducible nitric oxide synthase), and (or) 100 microM diethyldithiocarbamate (DETC, an inhibitor of Cu,Zn-SOD). In the KO macrophages, there was no protein band detected by Western blot with anti-GPX1 antibody and 98% reduction in total GPX activity compared with WT cells. Nitric oxide (NO) synthesis was greatly enhanced after 24 h by GPX1 knockout and DQ, but inhibited by AG or DETC. Protein carbonyl formation in total cell extract was clearly associated with NO synthesis as higher levels of protein carbonyl were detected in activated KO than WT macrophages, and DQ enhanced slightly while AG or DETC virtually blocked its formation. A similarly marginal effect of GPX1 KO was observed on protein nitration. The LPS/IFN/DQ-induced DNA fragmentation was blocked by AG, but not by DETC. Cell viability at 48 h was decreased by the LPS/IFN activation and further reduced by the addition of DQ, but restored by AG. In conclusion, GPX1 affects the NO production in activated peritoneal macrophages and protects these cells against NO-associated protein oxidation.

The Ontogeny and Induction by Zinc of Hepatic Chick Embryo Metallothionein

Abstract To avoid the maternal influences inherent in mammalian models, the chick embryo was examined for the effects of development on the ontogeny of hepatic metallothionein (MT) induction. Livers from embryos were examined for total and cytosolic zinc (Zn) and copper (Cu), and cytosolic MT on 12, 14, 16, and 18-21 days of incubation (DI) as well as 1, 7, and 14 days posthatch (ph). Cytosolic Zn levels fell, from a high on 12 DI (45.04 ± 1.05 ng/μg DNA), to 25.79 ± 1.05 ng/μg DNA on 19 DI and then increased to 44.73 ± 3.47 ng/μg DNA by 1 day ph. Cytosolic Cu followed a reverse pattern, with a high of 24.10 ± 0.74 ng/μg DNA on 16 DI. Both 109Cd radioassay analysis and gel filtration chromatography of hepatic cytosols showed a developmental pattern for MT similar to that of cytosolic Zn. These results are in contrast to the developmental pattern of the rat. To establish if yolk Zn levels limit hepatic MT in the 1-day neonate, the yolks of unincubated fertile eggs were supplemented with 26, 52, or 78% of the endogenous Zn (768 μg/yolk). As a result, hepatic MT of 1-day neonates increased by 3.9-, 4.7-, and 7.1-fold, respectively. Thus the initial level of Zn in the yolk has a significant impact on the final concentration of MT in neonatal liver. A study of MT induction in the 18-DI embryo revealed that yolk sac administration of Zn (550, 2750, 4950 μg/yolk) increased hepatic MT by 5.8-, 23-, and 39-fold, respectively. This demonstrates the inducibility of MT even at the point of minimum endogenous MT (18 DI). In summary, our results show that (1) a marked difference exists between the developmental patterns of MT in avian and mammalian species, and (2) chick embryo hepatic MT is highly responsive to exogenous Zn introduced into the yolk.

Iron-Induced Accumulation of Hepatic Metallothionein: The Lack of Glucocorticoid Involvement

Abstract The process(es) by which parenteral iron effects the accumulation of hepatic metallothionein (MT) is not known. The present study examined glucocorticoids as potential mediators of this process. Chicks were given either one injection (ip) of iron (+1FE) at 10 mg Fe/kg, two injections of iron (+2FE) given 24 hr apart, or a single injection of saline. Plasma corticosterone was evaluated at various times following the last injection. Plasma corticosterone increased approximately 50% following + 1FE but more than 200% at 2 and 4 hr following a second injection of iron (+2FE). Plasma zinc showed a transient increase followed by a considerable depression. Coincidentally, the accumulation (determined at 24 hr) of zinc MT in liver of +2FE chicks was three times higher than that of +1FE chicks. In another experiment, markedly greater changes, at similar time intervals, in plasma corticosterone were effected by multiple subcutaneous injections of adrenocorticotropic hormone (ACTH) (either 5 IU ACTH or 20 IU ACTH/kg). Subsequent analysis of hepatic zinc MT showed only minor changes as a result of ACTH injections. These results indicate that a change in the plasma glucocorticoid corticosterone is not a primary component in the processes) by which parenteral iron effects an increase in hepatic zinc MT.

Metalloforms of Metallothionein Induced by Parenteral Copper: The Influence of Route of Administration

Previous results regarding the two metalloforms of MT which accumulate in chick liver following the parenteral administration (ip) of copper were discussed. One metalloform, which is exclusively zinc, was suggested to reflect the marked accumulation of hepatic zinc following copper injection. The present report shows that there is a marked difference in hepatic zinc accumulation if copper is administered iv. Under these conditions there is virtually no change in hepatic zinc and thus MT produced under these conditions appears to contain only copper. We suggest the the changes in zinc metabolism as effected by copper when given intraperitoneally reflect a secondary response analogous to that observed when iron is similarly administered.

Parenteral zinc and tissue metallothionein in normal and diabetic rats

The effect of parenteral zinc on tissue metallothionein (MT) was studied in normal and streptozotocin-induced diabetic rats. The accumulation of Zn-MT in liver and pancreas of normal and diabetic rats following the administration (ip) of various amounts of zinc was not different. Renal Zn-MT was higher in the diabetic group, and this was not changed by zinc injection. Although diabetic rats, relative to normal, possessed a markedly higher concentration of Cu-MT in kidney initially, this difference decreased considerably after zinc injection. The ratio of Cu-MT to cytosolic Cu in kidney was not affected by parenteral zinc and was highest in diabetic rats. Zinc injection markedly reduced food intake, water consumption, and urine output in both normal and diabetic rats. Blood glucose of diabetic rats also decreased 24 h after zinc administration. Our results indicate that relative to normal, MT and zinc metabolism are different in kidney, and to some extent liver, but not different in the pancreas of the chemically induced diabetic rat.

Accumulation of Hepatic Zn7 and Zn/Cu Metallothionein in Copper-Loaded Chicks: Isolation and Characterization

The induction of metallothionein (MT) by copper has historically been a controversial area of research. Initial work in the mid 1970’s suggested that parenteral copper caused induction of a cysteine-rich protein (copper chelatin) distinct from MT. However, this protein is now considered to be an artifact.1