Liquid egg as an alternative protein source in calf milk replacers.

The use of alternative proteins in milk replacer has been evaluated for their ability to decrease the cost of milk replacers without negatively impacting performance of the calf. Three studies were conducted to evaluate the performance of calves fed milk replacer utilizing liquid egg as an alternative protein and to determine the optimal concentration of liquid egg to include in milk replacers. Calves in trials 1 and 2 were assigned to a control diet of all milk protein replacer (MILK) or a diet formulated to contain 5% of the diet (13.5% of the protein) from liquid egg (5% EGG). Calves in trial 3 were assigned to one of four diets: the control (MILK) and 5% EGG diets fed in trials 1 and 2, or diets formulated to contain either 10 or 15% of the diet (27 or 40.5% of the protein) from liquid egg (10% EGG, 15% EGG). For all experiments, milk replacers were formulated to contain 20% protein, 20% fat and were fed at 454 g/d reconstituted to 12% DM. Production of the diets containing egg protein utilized breaker eggs that were pasteurized during manufacturing. Holstein bull calves (n = 44 for experiment 1, n = 38 for experiment 2, and n = 120 for experiment 3), were purchased from an area sale barn. Calves were housed in individual hutches with water available free choice starting on d 0. A commercially available calf starter was offered free choice beginning on d 7 for experiments 1 and 2 and on d 1 for experiment 3. Feed intake, scour scores, and antibiotic treatments were recorded daily. For experiment 1, calves fed 5% EGG had greater weight gains than calves fed MILK. No differences in average daily feed intake were observed. For experiment 2, weight gains tended to be lower with 5% EGG, whereas feed intakes and gain to feed ratios were similar between calves fed MILK or 5% EGG. For experiment 3, as the amount of egg in the diet increased, weight gain decreased in a linear fashion during the milk replacer feeding period, but the decrease in gain was significant only with the 15% EGG diet. These results indicate that egg is an effective alternative protein source to milk protein in calf milk replacers when fed at levels up to 10% of the diet in a conventional feeding program of 0.45 kg per head per day.

Effects of selected polychlorinated biphenyl (PCB) congeners on hepatic glutathione, glutathione-related enzymes, and selenium status: implications for oxidative stress.

Polychlorinated biphenyls (PCBs) induce drug metabolism that may lead to the bioactivation of PCBs themselves or alternatively may lead to oxidative events within the cell. The goal of the present study was to determine the influence of congeneric PCBs, selected as substrates for or inducers of drug metabolism, upon hepatic glutathione, glutathione-related enzymes, and selenium status. Male and female Sprague-Dawley rats received two i.p. injections per week of PCB 3 (4-chlorobiphenyl), PCB 28 (2,4,4'-trichlorobiphenyl), PCB 38 (3,4,5-trichlorobiphenyl), PCB 77 (3,3',4,4'-tetrachlorobiphenyl), PCB 153 (2,2',4,4',5,5'-hexachlorobiphenyl), or both PCBs 77 and 153 (100 micromol/kg/injection) and were killed at the end of 1, 2, or 3 weeks. Whole liver homogenates, hepatic cytosol, and microsomes were prepared. Both glutathione reductase and glutathione transferase activities were increased significantly in both male and female rats receiving PCB 77, an aryl hydrocarbon receptor agonist, as well as in those receiving both PCBs 77 and 153. No significant trend was observed in the levels of hepatic total glutathione. PCB 77 treatment decreased hepatic selenium-dependent glutathione peroxidase (SeGPX) activity in both male and female rats significantly. This decrease in activity following PCB 77 treatment was accompanied by a decrease in the cytosolic selenium-dependent glutathione peroxidase gene (GSPx1) transcript, as well as a decrease in hepatic total selenium levels. These data support the concept that exposure to the coplanar PCB 77 suppresses, via gene regulatory mechanisms, the cellular antioxidant enzyme SeGPX and that this decrease involves selenium. Lower halogenated PCBs that may be bioactivated to reactive oxygen species (ROS)-producing metabolites, and higher halogenated PCBs that are not Ah receptor agonists, were inactive.

Relationships with serum parathyroid hormone in old institutionalized subjects.

OBJECTIVE AND BACKGROUND: Old people in residential care are at the highest risk of any group for hip fracture. This may relate to their high prevalence of hyperparathyroidism. There are few data, however, on relationships with serum parathyroid hormone (PTH) in these individuals. This study therefore examined complex associations with serum PTH in nursing home and hostel residents. DESIGN: Cross-sectional analysis. PATIENTS: One hundred and forty-three nursing home and hostel residents of median age 84 years. MEASUREMENTS: Serum PTH, 25-hydroxyvitamin D (25OHD), 1,25-dihydroxyvitamin D (1,25-(OH)2D), plasma creatinine, phosphate, calcium, albumin, Bsm-1 vitamin D receptor genotype, age, weight and use of frusemide or thiazide. RESULTS: The statistical models determined accounted for half the interindividual variation in serum PTH. Heavier weight was associated with both the prevalence of secondary hyperparathyroidism and the serum concentration of PTH. Novel interactions with serum PTH were identified between: weight and 25OHD; 25OHD and phosphate; and phosphate and thiazide diuretic use. Plasma phosphate was associated with PTH independently of calcium and 1,25-(OH)2D. There was no independent association between PTH and nuclear vitamin D receptor genotype. CONCLUSIONS: Heavier weight is associated with both the prevalence and severity of secondary hyperparathyroidism and consistent with animal models of secondary hyperparathyroidism, phosphate may relate to serum PTH independently of 1,25-(OH)2D or calcium.

Effects of peroxisome proliferators on antioxidant enzymes and antioxidant vitamins in rats and hamsters.

Peroxisome proliferators (PPs) cause hepatomegaly, peroxisome proliferation, and hepatocarcinogenesis in rats and mice, whereas hamsters are less responsive to PPs. PPs increase the activities of enzymes involved in peroxisomal beta-oxidation and omega-hydroxylation of fatty acids, which has been hypothesized to result in oxidative stress. The hypothesis of this study was that differential modulation of antioxidant enzymes and vitamins might account for differences in species susceptibility to PPs. Accordingly, we measured the activities of DT-diaphorase and superoxide dismutase (SOD) and the hepatic content of ascorbic acid and alpha-tocopherol in male Sprague-Dawley rats and Syrian hamsters fed 2 doses of 3 known peroxisome proliferators (dibutyl phthalate [DBP], gemfibrozil, and [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (Wy-14,643) for 6, 34, or 90 days. In untreated animals, the activity of DT-diaphorase was much higher in hamsters than in rats, but the control levels of SOD, ascorbic acid and alpha-tocopherol were similar. In rats and hamsters treated with Wy-14,643, we observed decreases in alpha-tocopherol content and total SOD activity. DT-diaphorase was decreased in activity following Wy-14,643 treatment in rats at all time points and doses, but only sporadically affected in hamsters. Rats and hamsters treated with DBP demonstrated increased SOD activity at 6 days; however, in the rat, DBP decreased SOD activity at 90 days and alpha-tocopherol content was decreased throughout. In gemfibrozil treated rats and hamsters, a decrease in alpha-tocopherol content and an increase in DT-diaphorase activity were observed. In either species, no consistent trend was observed in total ascorbic acid content after treatment with any of the PPs. In conclusion, these data suggest that both rats and hamsters are compromised in antioxidant capabilities following PP treatment and additional hypotheses for species susceptibility should be considered.

Polychlorinated biphenyl-induced effects on metabolic enzymes, AP-1 binding, vitamin E, and oxidative stress in the rat liver.

Environmental pollutants, such as polychlorinated biphenyls (PCBs), may induce drug metabolism and may be substrates for the induced metabolic enzymes. Both processes may lead to oxidative stress. The goal of this study was to determine the influence of polychlorinated biphenyls, selected as inducers and substrates of drug metabolism, on oxidative events within the liver over a 3-week time course. Male and female Sprague-Dawley rats received two ip injections per week of 4-chlorobiphenyl, 2,4,4'-trichlorobiphenyl, 3,4,5-trichlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl (PCB 77), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153), or both PCB 77 and 153 (100 micromol/kg/injection) and were euthanized at the end of 1, 2, or 3 weeks. Hepatic cytochrome P450 1A1 (EROD) activity, DT-diaphorase activity, AP-1 DNA-binding activity, conjugated dienes, and alpha-tocopherol (vitamin E) as well as alpha-tocopheryl quinone (oxidized vitamin E) were determined. While the lower chlorinated biphenyls (at these doses and times) showed little or no effect on these oxidative stress parameters, both CYP 1A1 and DT-diaphorase activities were significantly increased in both male and female rats receiving PCB 77, a ligand for the aryl hydrocarbon receptor. In addition, the DNA-binding activity of the transcription factor AP-1 was increased in rats treated with PCB 77 or PCB 153. Within the lipid fraction there was no significant increase observed in conjugated diene concentrations, but there was a significant increase in alpha-tocopheryl quinone upon treatment with all PCBs tested. These data indicate that alpha-tocopheryl quinone may be a sensitive marker for PCB exposure and is possibly increased by a wide range of PCBs.

Effects of peroxisome proliferators on glutathione and glutathione-related enzymes in rats and hamsters.

Peroxisomeproliferators (PPs) cause hepatomegaly, peroxisome proliferation, and hepatocarcinogenesis in rats and mice. Conversely, hamsters are less responsive to these compounds. PPs increase peroxisomal beta-oxidation and P4504A subfamily activity, which has been hypothesized to result in oxidative stress. We hypothesized that differential modulation of glutathione-related defenses could account for the resulting difference in species susceptibility following PP administration. Accordingly, we measured glutathione S-transferase (GST), glutathione peroxidase (GPx), and glutathione reductase (GR) activities, and total glutathione (GSH) in male Sprague-Dawley rats and Syrian hamsters fed two doses of three known peroxisome proliferators [dibutylphthalate (DBP), gemfibrozil, and Wy-14,643] for 6, 34, or 90 days. In rats, decreases in GR, GST, and selenium-dependent GPx were observed following PP treatment at various time points. In hamsters, we observed higher basal levels of activities for GR, GST, and selenium-dependent GPx compared to rats. In addition, hamsters showed decreases in GR and GST activities following PP treatment. Interestingly, selenium-dependent GPx activity was increased in hamsters following treatment with Wy-14,643 and DBP. Treatment for 90 days with Wy-14,643 resulted in no change in GPx1 mRNA in rats and increased GPx1 mRNA in hamsters. Sporadic changes in total GSH and selenium-independent GPx were observed in both species. This divergence in the hydrogen peroxide detoxification ability between rats and hamsters could be a contributing factor in the proposed oxidative stress mechanism of PPs observed in responsive and nonresponsive species.

Cellular response to a glutathione S-transferase P1-1 activated prodrug.

TER286 [gamma-glutamyl-alpha-amino-beta(2-ethyl-N,N,N', N'-tetrakis(2-chloroethyl)phosphorodiamidate)-sulfonyl-propionyl-( R)- (-) phenylglycine] is a novel nitrogen mustard prodrug that is preferentially activated by glutathione S-transferase P1-1 (GSTP1-1). A human promyelocytic leukemia /TER286-resistant cell line was selected by chronic, long-term exposure to the prodrug. Although resistance was not readily achieved, eventually a 5-fold resistant clone was isolated. Cross-resistance to melphalan occurred, but not to doxorubicin (Adriamycin), taxol, and gamma-glutamyl-S-(benzyl)cysteinyl-R(-)-phenyl glycine diethyl ester, a GSTP1-1 inhibitor. The protein and transcript levels and enzymatic activity of GSTP1-1 were reduced significantly in the selected resistant line. GSTalpha levels were unchanged, and GSTmu was undetectable. Although glutathione levels were elevated in human promyelocytic leukemia/TER286 cells, no changes in the expression of thiol-related genes including gamma-glutamylcysteine synthetase, gamma-glutamyl transpeptidase, or multidrug resistance protein were found. A 7-fold increase in catalase expression in the resistant cell line indicated an adaptive response to oxidative and electrophilic stress, and this was also reflected in the lower prevalence of drug-induced DNA single-strand breaks in the resistant cells. Mouse embryo fibroblast GSTP1-1(-/-) cells exhibited 2-fold resistance to TER286 compared with GSTP1-1(+/+) cells. NIH3T3 cells transfected with combinations of gamma-GCS and multidrug resistance protein exhibited enhanced resistance to TER286, although the degree of resistance was impaired by cotransfection of GSTP1-1. These results are consistent with responses in the TER286-resistant cells indicative of GSTP1-1-mediated mechanism of activation. In consequence, these data support the rationale that tumors expressing high levels of GSTP1-1 will be more sensitive to the cytotoxic effects of the drug.

Glutathione peptidomimetic drug modulator of multidrug resistance-associated protein.

The peptidomimetic drug gamma-glutamyl-S-(benzyl)cysteinyl-R-(-)-phenyl glycine diethyl ester (TER199) is an analog of glutathione designed to be an isozyme-specific inhibitor of GSTP1-1 protein1-1. This compound (and the de-esterified moiety) is shown to be an effective inhibitor of multidrug resistance-associated protein1 (MRP1)-mediated drug resistance. Kinetic analyses revealed that gamma-glutamyl-S-(benzyl)cysteinyl-R-(-)-phenyl glycine reversibly inhibits the transport of 2,4-dinitrophenyl-S-glutathione with a K(i) of 752 microM. TER199 reversed the accumulation deficit of daunorubicin in MRP1-transfected NIH3T3 fibroblasts and maintained intracellular levels for >2 h after daunorubicin removal. Cytotoxicity assays revealed that TER199 significantly reversed the resistance of MRP1-transfected NIH3T3 cells for vincristine, doxorubicin, etoposide, and mitoxantrone. HL-60 cells made resistant to TER199 by chronic, long-term selection had increased mRNA and protein levels of multidrug resistance-associated protein, MRP1, and gamma-glutamyl cysteine synthetase heavy and light subunits (the rate-limiting enzyme in GSH synthesis). In spite of increased gamma-glutamyl cysteine synthetase, their glutathione content was reduced approximately 35% from that of parental HL-60 cells. These cells also exhibited a drug resistance profile commensurate with the previously described MRP1 overexpressing phenotype, with resistance to Vinca alkaloids, epipodophyllotoxins, and anthracyclines; additional cross-resistance to paclitaxel (Taxol), mitoxantrone, and 5-fluorouracil was observed.

Characterization of estrogen receptor-beta (ERbeta) messenger ribonucleic acid and protein expression in rat granulosa cells.

We have examined estrogen-responsiveness of ovarian granulosa cells by focusing on estrogen receptor (ER) expression. Estrogen responsiveness was determined by examining the effect of 17beta-estradiol (1-10 nM) on luciferase reporter activity in rat granulosa cells transfected with an ERE-luciferase construct. The results demonstrate an estrogen-induced (approximately 3-fold) increase in luciferase reporter activity, indicating that granulosa cells contain functional estrogen response element (ERE)-binding transcriptional activators. Gel mobility shift assays in combination with ER antibodies show that ERbeta is the predominant ERE-binding protein in granulosa cells. Western blotting results show that granulosa cells contain ERbeta-immunoreactive protein(s) migrating at a size substantially larger than the recombinant protein generated from the originally proposed 485 amino acid open-reading frame. This size discrepancy is not due to granulosa cell expression of ERbeta isoforms with insertions within the coding region because RT-PCR assays revealed products with sizes expected for ERbeta, ERbetaB, and delta3 isoforms. This size discrepancy appears to be due to usage of a well-conserved, upstream in-frame translation initiation codon (ATG436) leading to a 530 amino acid open reading frame. ERbeta messenger RNA (mRNA) characterization using 5'-rapid amplification of complementary DNA ends (5'-RACE) show the presence of two different (P1- and P2-) 5'-ends of rat ERbeta mRNA encoding the full-length ERbeta protein. The generation of the P2-specific exon is likely due to initiation of transcription from an alternative promoter. Both P1- and P2-specific exon-containing ERbeta mRNAs are expressed in granulosa cells, and they are rapidly down-regulated by the cAMP-mediated intracellular signaling pathway in cultured granulosa cells. Taken together, our results show that rat granulosa cells produce two different 3',5'-cAMP-regulated ERbeta mRNA species and that these mRNA species are capable of encoding the full-length ERbeta protein.

Regulation of protein kinase C delta by estrogen in the MCF-7 human breast cancer cell line.

We have previously shown that estrogen up-regulates expression of protein kinase C (PKC) delta in the rat and rabbit corpus luteum as well as in luteinized rat granulosa primary cell cultures. To determine whether a similar regulation of the PKC delta isoform by estrogen occurred in another estrogen responsive system, we investigated the estrogen receptor positive MCF-7 human breast cancer cells. In a characterization of PKC isoforms in MCF-7 cells we determined that PKC delta was the predominant PKC isoform. However in contrast to the effect of estrogen on PKC delta expression in ovarian cells, estrogen treatment of MCF-7 cells resulted in a significant decrease in PKC delta protein and mRNA expression in a time and dose dependent manner. Treatment of MCF-7 cells with 10(-10)-10(-8) M estrogen for 7 days down-regulated specifically PKC delta mRNA and protein while expression of other PKC isoforms was unchanged. The opposite regulation of PKC delta expression in ovarian and breast cancer cells prompted us to evaluate the type of estrogen receptor present in both cell types. Results showed that luteinized rat granulosa cells expressed predominantly estrogen receptor beta while the MCF-7 cells expressed predominantly estrogen receptor alpha and barely detectable levels of estrogen receptor beta. These results suggest that the differential ability of estrogen to regulate PKC beta expression could potentially be a result of differential signaling through the two estrogen receptor subtypes.

Regulation of the rat liver sodium-dependent bile acid cotransporter gene by prolactin. Mediation of transcriptional activation by Stat5.

The intracellular mechanism(s) underlying the upregulation of the hepatic Na+/taurocholate cotransporting polypeptide (ntcp) by prolactin (PRL) are unknown. In this report, we demonstrate a time-dependent increase in nuclear translocation of phosphorylated liver Stat5 (a member of the ignal ransducers and ctivators of ranscription family) that correlated with suckling-induced increases in serum PRL levels. In electrophoretic mobility gel shift assays, nuclear Stat5 exhibited specific DNA-binding ability towards IFN-gamma-activated sequence (GAS)-like elements (GLEs; 5'TTC/A-PyNPu-G/TAA-3') located in the -937 to -904 bp region of the ntcp promoter. Transient cotransfections in HepG2 cells revealed that PRL inducibility (2.5-3-fold) required coexpression of the long form of the PRL receptor (PRLRL) and Stat5. Deletion analysis mapped the PRLinducible region to -1237 to -758 bp of the ntcp promoter. Linking this 0.5-kb region to a heterologous thymidine kinase (tk) promoter, or linking multimerized ntcp GLEs either upstream of the ntcp minimal promoter (-158 to +47 bp) or the heterologous promoter conferred dose-dependent PRL responsiveness. The short form of the PRL receptor failed to transactivate ntcp GLEs. These results indicate that PRL acts via the PRLRL to facilitate Stat5 binding to ntcp-GLEs and to transcriptionally regulate ntcp.

Stereoselective effects of chiral clofibric acid analogs on rat peroxisome proliferator-activated receptor alpha (rPPAR alpha) activation and peroxisomal fatty acid beta-oxidation.

Enantiomers of a series of substituted analogs of 2-(4-chlorophenoxy) -acetic acid (CPAA) were synthesized and used to examine the influence of steric and structural parameters on peroxisome proliferation. The effects of these compounds were studied on the activation of the peroxisome proliferator-activated receptor alpha (PPAR alpha) in CV-1 cells using an in vitro co-transfection assay. Selected sets of isomers were tested for their ability to increase peroxisomal fatty acyl-CoA oxidase (ACO) activity in H4IIEC3 (rat Reuber hepatoma) cells. Of the series of 2-substituted analogs studied, the isomers of the nu-propyl and phenyl derivatives of CPAA showed a high degree of stereoselectivity [(S)-isomer >> (R)-isomer]. In general, the potency of the compound to activate the receptor increased with the size of the 2-alkyl substituent. Among the 4-chlorobenzyloxy- and 4-(4'-chlorophenyl)benzyloxy- analogs studied, 2-[4-(4'-chlorophenyl)-benzyloxy]-propanoic acid exhibited a high degree of stereoselectivity in both the biological systems studied [(R) >> (S)]. The congeners of 2-methyl substituted CPAA showed a reverse stereoselectivity (R) > (S)] as compared to the other 2-substituted analogs [(S) > (R)]. Our results indicate that (1) both structural and steric characteristics of CPAA analogs play an important role in the activation of rPPAR alpha and on stimulation of peroxisomal ACO activities, and (2) clofibric acid and analogs exert their peroxisome proliferative effects by interaction with a specific site on a protein. The enantiomers of the 2-nu-propyl and the 2-phenyl CPAA analogs may be useful as mechanistic probes in elucidating the nature of this binding site.

Convergence of three steroid receptor pathways in the mediation of nongenotoxic hepatocarcinogenesis.

The mechanisms by which peroxisome proliferators are able to regulate metabolic processes such as fat metabolism, while at the same time creating an environment for the development of hepatocellular carcinomas, is a central issue in the non-genotoxic carcinogenesis field. The convergence of two members of the steroid receptor family (peroxisome proliferator-activated receptor, PPAR; and retinoid X receptor, RXR) has provided strong support for an oxidative stress component in this carcinogenesis process, but has yet to define clearly a pathway for the classical tumor promotion events associated with peroxisome proliferation. The findings presented here integrate a third member of the steroid receptor family into this process and suggest a novel autocrine loop and mechanism for creating both oxidative stress and tumor promotion. A central regulatory component in this pathway is farnesol which has recently been shown to induce transcription mediated by the steroid receptor family member, farnesoid X receptor (FXR). In this report, it is clearly demonstrated that farnesol can also upregulate the transcriptional events of PPAR, but most likely through a different farnesoid metabolite, resulting in the regulation of an entirely different set of genetic components. Deregulation of the activities of these receptors offers a provocative mechanism for explaining the hepatocarcinogenic effects of peroxisome proliferators in chronically treated rodents.

Peroxisome proliferator-activated receptor response specificities as defined in yeast and mammalian cell transcription assays.

Peroxisome proliferators include a heterogeneous group of xenobiotic agents capable of inducing peroxisome proliferation and hepatocellular carcinomas in rodent model systems. These chemicals appear to mediate their activity through a family of transcription factors known as peroxisome proliferator-activated receptors (PPAR). Recently it has been shown that DNA binding of PPAR is contingent upon heterodimerization with a member of the retinoic acid X (RXR) family of receptors. In this report transcription parameters of a rat PPAR alpha were analyzed using mammalian and yeast cotransfection assays. PPAR activity was observed to be peroxisome proliferator dependent in the mammalian cotransfection assay, and heterodimer dependent but peroxisome proliferator independent in a yeast version of the same assay. Moreover, when the naturally occurring ligand for RXR, 9-cis-retinoic acid (RA), was tested in the same assays, it was observed to generate an RXR-specific response in the yeast cell assay but host cell-specific response in the mammalian cell assay. Finally, the combination of peroxisome proliferator and 9-cis-RA had very little added effect on the yeast cell assay but again produced a cell-specific synergistic response in the mammalian cell assay. These data demonstrate that PPAR transcriptional activity is strongly influenced by the RXR family of receptors, and that peroxisome proliferators may be regulating PPAR mammalian cell activity through a secondary mechanism.