Alterations in immune function and CYP450 activity in adult male deer mice (Peromyscus maniculatus) following exposure to benzo[a]pyrene, pyrene, or chrysene.

Polycyclic aromatic hydrocarbons (PAHs) are among the most common classes of chemical contaminants found at hazardous waste sites. Deer mice (Peromyscus maniculatus) exhibit a wide geographic distribution throughout North America and have been suggested as a terrestrial biomonitoring species to facilitate comparisons between superfund sites. Chemicals tested were benzo[a]pyrene (BaP; CAS number 50-32-8), pyrene (Pyr; CAS number 129-00-0), and chrysene (Chr; CAS number 218-01-9). Adult male deer mice were exposed via intraperitoneal (i.p.) injection every other day for 11 d to the PAHs (0.3, 1, 3, 10, or 30 mg/kg) or a corn oil carrier control. Both BaP and Chr suppressed the plaque-forming cell (PFC) response at all treatment levels. Pyr exposure (1-30 mg/kg) also resulted in suppression of this response. Macrophage pinocytosis was suppressed only by Chr (3, 10, and 30 mg/kg). Concanavalin A-induced proliferation was stimulated by BaP at all dose levels, by Pyr at 1-30 mg/kg, and by Chr at 30 mg/kg. Chr did not affect pokeweed mitogen (PWM)-induced proliferation; however, BaP (1-30 mg/kg) and Pyr (0.3-30 mg/kg) produced stimulation of this response as compared to respective controls. BaP and Chr stimulated cytochrome P-450 1A1 (CYP1A1) activity (3, 10, or 30 mg/kg) as measured by ethoxyresorufin O-deethylase (EROD) activity, but Pyr did not. These results indicate that immune function endpoints appear to be more sensitive to these PAHs than measured hepatic CYP450 activity.

The effects of fumonisin B1 on viability and mitogenic response of avian immune cells.

Fumonisin B1 (FB1) is a mycotoxin produced by the fungus Fusarium verticillioides (formerly Fusarium moniliforme) and is found in diverse crops such as corn, wheat, and barley. Many diseases linked to FB1, such as porcine pulmonary edema, rat hepatic cancer, and equine leukoencephalomalacia, indicate a compromised immune system. The purpose of this study was to determine whether FB1 altered immunological responses in various cell populations of Single Comb White Leghorn chicks. Cells collected for this study were obtained from those immunological organs with well-defined responses (i.e., spleen, thymus, and blood). Cell populations were exposed to 5 to 50 microg/mL FB1 in vitro for 24 to 72 h, and viability and mitogenic response were evaluated. The effects of FB1 on the mitogenic response were evaluated in cell populations from the spleen and blood stimulated with the mitogens, lipopolysaccharide, concanavalin A, and pokeweed mitogen and in thymocytes stimulated with concanavalin A. The 3-(4,5-dimethylthazol-2-yl)-diphenyl-2H-tetrazolium bromide (MTT) reduction assay was used to assess viability and mitogenic response. Fumonisin B1 decreased spleen cell viability and mitogenic response, albeit the degree of decrease varied with mitogen and time of exposure. Fumonisin B1 increased number of viable thymic cells at 50 microg/mL but had no effect on the mitogenic response of thymocytes. Fumonisin B1 had no effect on blood lymphocyte viability or mitogenic response.

Elasmobranch immune cells as a source of novel tumor cell inhibitors: Implications for public health.

SYNOPSIS: Reports that elasmobranchs (sharks, skates, and rays) may have a low incidence of disease have stimulated interest in understanding the role of their immune system in this apparent resistance. Although research in this area may potentially translate into applications for human health, a basic understanding of the elasmobranch immune system components and how they function is essential. As in higher vertebrates, elasmobranch fishes possess thymus and spleen, but in the absence of bone marrow and lymph nodes, these fish have evolved unique lymphomyeloid tissues, namely epigonal and Leydig organs. As conditions for short-term culture of elasmobranch immune cells have become better understood, the opportunity to examine functional activity of cytokine-like factors derived from conditioned culture medium has resulted in the identification of growth inhibitory activity against a variety of tumor cell lines. Specifically, the medium enriched by short term culture of bonnethead shark (Sphyrna tiburo) epigonal cells (epigonal conditioned medium, ECM) has been shown to inhibit the growth of mammalian tumor cell lines, including fibrosarcoma (WEHI-164), melanoma (A375.S2), B-cell lymphoma (Daudi), T-cell leukemia (Jurkat), pancreatic cancer (PANC-1), ovarian cancer (NIH:OVCAR-3), and three breast carcinoma cell lines (MCF7, HCC38, Hs578T). Of the cell lines tested, WEHI-164, A375.S2, Daudi, and Jurkat cells were among the most sensitive to growth inhibitory activity of ECM whereas PANC-1 and NIH:OVCAR-3 cells were among the least sensitive. In addition, ECM demonstrated preferential growth inhibition of malignant cells in assays against two different malignant/non-malignant cell line pairs (HCC38/HCC38 BL and Hs 578T/Hs 578Bst). Separation of protein components of ECM using SDS-PAGE resulted in a very reproducible pattern of three major bands corresponding to molecular sizes of approximately 40-42 kD, 24 kD, and 17 kD. Activity is lost after heating at 75 degrees C for 30 min, and can be diminished by treatment with proteinase K and protease. Activity is not affected by treating with trypsin, DNase I or RNase A.

Dexamethasone-induced apoptosis in immune cells from peripheral circulation and lymphomyeloid tissues of juvenile clearnose skates, Raja eglanteria.

Juvenile clearnose skates (Raja eglanteria) were injected intramuscularly with dexamethasone-21-phosphate at 50, 75, and 100mg/kg body weight. After 24h, skates were sacrificed and lymphomyeloid tissues (thymus, spleen, Leydig organ, and epigonal organ) were removed and whole blood was sampled. Tissues were used fresh for imprints or prepared for histology by solvent fixation or freezing in liquid nitrogen. Apoptosis in fixed tissues was assessed by transmission electron microscopy. Frozen sections and cytospin preparations of peripheral blood leukocytes (PBL) were evaluated by the TUNEL reaction to detect DNA strand breaks. Dexamethasone treatment increased apoptotic activity in all lymphomyeloid tissues as well as in PBL. These studies demonstrate that immune cells of elasmobranchs have the capacity for glucocorticoid-driven apoptosis, and that programmed cell death as a mechanism to regulate immune cell production appears to have been conserved during vertebrate evolution.

Seminal plasma and IVF potential. Biochemical constituents of seminal plasma of males from in vitro fertilization couples.

PURPOSE: Many components of seminal plasma play a role in sperm motility by serving as energy sources. Human seminal plasma contains over 30 proteins, including forward motility proteins, antifertility proteins, and coagulation/liquefaction proteins. This study was designed to determine any correlation between motility or fertilization rates and concentrations of fructose, lactic acid, citric acid, carnitine, and protein in human seminal plasma. METHODS: Fertilization rates were determined by in vitro methods. Fructose, lactic acid, citric acid, and carnitine concentrations were ascertained using high performance liquid chromatography. Protein concentration was determined by Bradford assay. RESULTS: Protein concentrations were significantly different as a function of sperm motility levels. Other constituents of human seminal plasma showed an overall correlation, though not significant. No constituent exhibited significant differences as a function of fertility levels. CONCLUSIONS: Protein concentration was significantly lower for samples with high motility. No significant differences between fertility levels and constituents measured were found.

Removal of dairy soil from heated stainless steel surfaces: use of ozonated water as a prerinse.

Square (2.54 x 2.54 cm2) 304 stainless steel metal plates were cleaned, passivated, and soiled by autoclaving (121 degrees C at 15 psi for 15 min) with reconstituted nonfat dry milk (20% solids). Fifteen-minute treatments using either warm water (40 degrees C) or ozonated cold water (10 degrees C) were conducted to compare prerinse cleaning potential of soiled metal plates. The chemical oxygen demand determination was performed on extracted organic material from treated metal plates. Results indicated that the ozone treatment removed 84% of soil from metal plates versus 51% soil removal by the warm water treatment, but the effectiveness of the two treatments did not differ (P > 0.05). Cleaning effects were visualized using scanning electron microscopy at 200x and 2000x magnification. The amount of soil film present on stainless steel metal surfaces was visibly lower on ozonated treatments versus on warm water treatments.

Expression of epidermal growth factor (EGF) and the EGF receptor in the porcine oviduct.

The production, secretion, and localization of epidermal growth factor (EGF) and the distribution of the EGF receptor (EGF-R) were examined in the isthmus (I) and ampulla (A) of the oviducts from cyclic (C) and early-pregnant (P) gilts. Sexually mature gilts (n = 20) were divided equally into two groups: C and P. P gilts were bred twice (at 0 and 24 h), and all gilts were killed 48 h after onset of estrus. After removal of reproductive tracts, oviducts were isolated, flushed, opened longitudinally, divided by anatomical region, cut into 1-3-mm3 pieces, and placed in Dulbecco's modified Eagle's Essential medium (DMEM: F-12 + ITS [insulin, 5 micrograms/ml; transferrin, 5 micrograms/ml; and selenious acid, 5 ng/ml] + antibiotic). Half the tissue and medium were immediately homogenized and centrifuged, and the supernatant was removed. The remaining tissue was cultured in the medium for 24 h at 37 degrees C and 5% CO2, then prepared similarly for analysis. EGF was measured in the supernatant by a heterologous RIA. Concentration of EGF was expressed as nanogram/milliliter of EGF per milligram of protein in wet tissue. EGF concentrations were present in both regions of the oviducts of C and P gilts. It was greater in I than in A tissues for both C (I = 16.21 ng/ml vs. A = 13.91 ng/ml; p < 0.05) and P gilts (I = 14.27 ng/ml vs. A = 12.53 ng/ml; p < 0.10). Higher concentrations of EGF were found in I tissue of C gilts than in P gilts (C = 16.21 ng/ml vs. P = 14.27 ng/ml; p < 0.05). The media assayed from cultured explants of I and A sections from C and P gilts gave results that were highly correlated with those of immediately prepared tissue sections. Localization of EGF in frozen oviductal tissue sections was demonstrated by immunohistochemistry. The primary site of EGF immunostaining occurred in the epithelial cells (with highest intensity at the apical border) of both C and P gilts. A and I tissue sections from C gilts showed localization of EGF immunostaining mainly in epithelial cells and lamina propria cells, while those from P gilts stained less intensely. The presence of EGF-R was shown by incubating tissue imprints and frozen sections with EGF-erythrosin isothiocyanate, which revealed that EGF-R were distributed mainly on the membranes of epithelial cells. The study indicates that EGF and EGF-R are present in oviductal epithelial cells in both C and P gilts, with the highest concentration of EGF in C gilts.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and purification of a protein that induces production of the Lactobacillus acidophilus bacteriocin lactacin B.

Lactacin B is a heat-stable bacteriocin produced by Lactobacillus acidophilus N2 that is active against closely related lactobacilli, including Lactobacillus delbrueckii subsp. lactis (formerly Lactobacillus leichmannii) ATCC 4797. Pure producer cultures propagated in MRS broth (initial pH 6.5) contain no lactacin B; it is detected only in cultures maintained at pH 5.0 to 6.0 and produced optimally at pH 6.0 S. F. Barefoot and T. R. Klaenhammer, Antimicrob. Agents Chemother. 26:328-334, 1984). Associative growth of producer and indicator, L. delbrueckii subsp. lactis ATCC 4797, resulted in production of an inhibitor identical to lactacin B. Associative growth increased lactacin B production from nondetectable levels (< 100 activity units [AU]/ml) to between 3,200 and 6,400 AU/ml in MRS broth (initial pH 6.5) and resulted in early but equal production of lactacin B (approximately 25,600 AU/ml) in broth maintained at pH 6.0. Indicator cells, but not spent culture filtrates, induced lactacin B production. Indicator cells disrupted by a French pressure cell yielded cell-free filtrates containing inducing activity. Chromatofocusing and gel filtration high-performance liquid chromatography of cell-free filtrates yielded a protein with a pI of 4.1 and a molecular size of approximately 58 kDa that induced lactacin B production. Analytical isoelectric focusing yielded a single protein band. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels contained a 28-kDa protein suggesting a two-subunit structure. Protein sequencing identified an N-terminal serine and 18 additional amino acids. To our knowledge, there are not previous descriptions of proteins that induce bacteriocin production in lactic acid bacteria.

Proteolytic enzymes in seminal plasma of domestic turkey (Meleagris gallopavo).

Protease and basic amidase activity was found in the seminal plasma of the domestic turkey. Amidase activity, measured through use of N-alpha-benzoyl-DL-arginine-p-nitroanilide-HCL (BAPNA), was 23-28 times greater for turkey than for guinea fowl or chicken. Within the reproductive tract, seminal plasma from the vas deferens had much greater activity than testicular or epididymal fluids. Turkey seminal plasma enzyme (TSPE) purified by chromatography or isoelectric focusing showed three protein bands by PAGE, each resolving on SDS-PAGE into two subunits with molecular weights of approximately 28,000-32,000 and 38,000-44,000. One of the three proteins also contained a larger subunit (M(r) 76,000-81,000) thought to be transferrin. Turkey acrosin consisted of three subunits with molecular weights below 20,500. Acrosin, but not TSPE, was visualized in native gels with N-alpha-benzoyl-DL-arginine-beta-naphthylamide (BANA)/Fast Garnet stain. Michaelis constants (BAPNA) for TSPE, acrosin, and trypsin were 2.41 +/- 0.12 x 10(-4) M (n = 5), 4.96-6.03 x 10(-4) M (n = 2), and 6.76 +/- 0.95 x 10(-4) M (n = 6), respectively. TSPE, like acrosin and trypsin, was inhibited by benzamidine but not iodoacetamide. While all natural trypsin inhibitors tested inhibited acrosin, TSPE was not inhibited by ovomucoid from chicken or turkey egg white.

Research note: kinetic and inhibition studies with turkey acrosin.

Acrosin was extracted from turkey spermatozoa and partially purified by chromatofocusing. Enzyme activity was tested over a pH range with three different substrates. In each case, the pH optimum was between pH 8 and 9. When N-alpha-benzoyl-DL-arginine-p-nitroanilide.HCl (BAPNA) was used as a substrate, the Km and Vmax were 1.17 +/- .05 x 10(-3) M and 1.50 +/- .07 x 10(4) mumol/min.milligram, respectively. Turkey acrosin amidase activity was inhibited by aprotinin, ovomucoid, soybean trypsin inhibitor, benzamidine, p-aminobenzamidine, and zinc.

Genetic selection for aflatoxin B1 resistance influences chicken T-cell and thymocyte proliferation.

Studies were conducted with two lines of chickens that were selected for high and low plasma protein concentrations in response to aflatoxin B1 (AFB1) exposure. The experiments were designed to determine genetic differences in the responses of T cells and thymocytes to the toxin. Chicks were orally administered AFB1 at a rate of 0, 100, or 500 micrograms/kg body weight up to 21 days of age. At 4 weeks of age, concanavalin A (Con A, 2.5 micrograms/mL) stimulated T-cell proliferation was similar for untreated chicks from the low line (LL) and the high line (HL). However, AFB1 reduced the responses of T cells with HL cells being more sensitive. In a second experiment, immature chickens were bled and peripheral blood lymphocytes were cultured with Con A and either 0, 3.125, 6.25, 12.5, or 25 micrograms/mL AFB1. T cells from LL had greater responses to Con A than those from HL, and LL T-cells were also more resistant to in vitro AFB1 exposure. Furthermore, thymocyte proliferation was greater for LL chicks; but when thymocytes were cultured with 25 micrograms/mL AFB1, 3H-thymidine incorporation was similarly reduced in both lines. Cell cycle analysis indicated that there were more LL thymocytes in S phase, and the percentages for both lines decreased with AFB1 treatment. Although there were no differences between the lines for percent G2/M cells, AFB1 treatment increased the percentages of thymocytes in G2/M. These studies showed that selection for plasma protein response also changed T-cell and thymocyte proliferative activity.

In vitro metabolism of the pro-carcinogen aflatoxin B1 by liver preparations of the calf, nurse shark and clearnose skate.

1. Liver postmitochondrial supernatant preparations of calf, clearnose skate, and nurse shark were able to metabolize the fungal toxin aflatoxin B1 to various metabolites. 2. Calf liver produced aflatoxin M1 and Q1 as the major chloroform soluble metabolites, with small amounts of aflatoxicol formed during incubation. 3. Liver preparations of the elasmobranchs, however, produced aflatoxicol as the major chloroform soluble metabolite with no other metabolite being detected. 4. The water soluble metabolite profiles for the three species were also quite different with the tris diol adduct being produced to a much greater extent in calf liver preparations. 5. Aflatoxicol production by the elasmobranch liver homogenates was reversible with the skate reconverting a large amount (30%) of aflatoxicol to AFB1. The nurse shark, however, appeared to convert a portion of aflatoxicol to an unknown metabolite more polar than AFB1. 6. Calf liver DNA bound approximately 3 x more 3H-AFB1 than shark liver DNA.

Turkey acrosin. I. Isolation, purification, and partial characterization.

Acrosin was extracted from turkey spermatozoa by use of urea together with sonication and freezing, and purified approximately 18-fold by sequential use of chromatofocusing and affinity chromatography. The use of chromatofocusing for the initial purification step proved to be superior to preparative isoelectric focusing. Similar to acrosin from many mammalian species, turkey acrosin was found to be a glycoprotein possessing characteristics of serine proteases. Polyacrylamide gel electrophoresis (PAGE) of the enzyme indicated the presence of two isozymes. Sodium-dodecyl sulfate PAGE under reducing conditions revealed three subunits with approximate molecular weights of 11,700, 13,900, and 15,900.

Quality assessment of pasteurized fluid milk as related to lipopolysaccharide content.

A study was conducted to investigate any relationship between lipopolysaccharide (endotoxin) concentration in milk and parameters associated with the keeping quality of milk. Parameters investigated were flavor intensity, standard plate count, coliform count, psychrotrophic count, and gram-negative count. Lipopolysaccharide content was determined using the Limulus Amoebocyte Lysate assay. Pasteurized whole milk samples were obtained the day of processing in .94-L containers with samples analyzed on d 0 through 21. Significant linear relationships were detected between lipopolysaccharide concentration and days storage at 7 degrees C, flavor intensity, and psychrotrophic count.

Quantitative assay for antibiotics used commonly in treatment of bovine infections.

Delvotest-P and Bacillus stearothermophilus Difco disc assay procedures were utilized, and assay sensitivity for detecting various antibiotics was tested. Bacillus stearothermophilus spores used in the disc assay were purchased from two laboratories and compared. The dyes methylene blue, 2,3,5-triphenyltetrazolium chloride, orange-O, and bromcresol purple were incorporated separately into agar used for the disc assay to determine if sharper, clearer zones could be produced. None of the dyes had an observable effect on zone size or clarity. Using the Difco disc assay, quantitation of penicillin, cephapirin, and oxytetracycline was accomplished with reliable precision by relating the size of the zone of inhibition to the log of the antibiotic concentration. The Delvotest-P and Difco disc assays were equal in sensitivity, as were spore suspensions used from the two laboratories.

A comparative study of monooxygenase activity in elasmobranchs and mammals: activation of the model pro-carcinogen aflatoxin B1 by liver preparations of calf, nurse shark and clearnose skate.

Liver microsome preparations of the elasmobranchs Ginglymostoma cirratum (nurse shark) and Raja eglanteria (clearnose skate) were examined for monooxygenase activity using aflatoxin B1 as substrate. At equiprotein concentrations, elasmobranch microsomes were less than 20% as active as calf liver in producing mutagenic metabolites of aflatoxin B1.

Effects of aflatoxin M1 intake at physiologic levels on newborn dairy calves.

When aflatoxin-contaminated grain is consumed by dairy cows, aflatoxin M1 is excreted in the milk. Sixteen neonatal male Holstein calves were given milk which had been collected from cows given 5 to 6 mg of aflatoxin B1 each day. The calves were examined for possible detrimental effects of the mycotoxin at pseudophysiologic concentrations. Calves were allotted to 1 of 4 groups given different milk dietary aflatoxin M1 concentrations: group 1--given 0 microgram of aflatoxin M1/L (undetectable); group 2--given 0.5 microgram/L; group 3--given 1 microgram/L; and group 4--given 2 micrograms/L. Whole milk equal to 8% of body weight was fed daily and adjusted each week to maintain this ratio. Water and a 15% crude protein complete calf starter ration were offered ad libitum for the 6-week feeding study. Weekly blood samples were collected via jugular venipuncture and analyzed for serum alkaline phosphatase and aspartate aminotransferase activities. Daily means for milk dry matter intake (in kg) and complete ration intake (in kg) for the calf groups were as follows: 0.46 and 0.36 for group 1; 0.46 and 0.25 for group 2; 0.42 and 0.18 for group 3; and 0.49 and 0.40 for group 4. Significant differences in complete ration and total dry matter intake were noted. The average daily gains (in kg) and gains in height at withers (in cm) were 0.39 and 4.1 for group 1; 0.36 and 4.0 for group 2; 0.29 and 5.7 for group 3; and 0.42 and 5.1 for group 4.(ABSTRACT TRUNCATED AT 250 WORDS)

Retention data for antibiotics commonly used for bovine infections.

This study was designed to observe the maximum time that various antibiotics used in different ways as treatment of bovine infections persisted in milk after final treatment. Both Delvotest-P and Bacillus stearothermophilus (Difco) disc assay procedures were utilized for detection of antibiotic preparations used for treatment of mastitis. None persisted in milk longer than specified on their respective labels. Because antibiotic residues were detected in milk consequent to treatment for intrauterine infections, guidelines for withholding times following intrauterine treatment should be established.

Effect of aflatoxin B1 and major metabolites on phytohemeagglutinin-stimulated lymphoblastogenesis of bovine lymphocytes.

Effects of aflatoxin B1 and three of its metabolites on cellular immune response were assessed with an assay based on inhibition of tritiated thymidine uptake by phytohemeagglutinin stimulated lymphocytes. In this in vitro system aflatoxin B1 and aflatoxin Q1 were strongly inhibitory (more than 50% inhibition) at concentrations of 10 mu/ml, whereas aflatoxicol and aflatoxin B2 alpha exhibited little inhibition at 10 micrograms/ml and only 45 to 50% inhibition at 25 micrograms/ml. Contrasts with single degrees of freedom and orthogonal polynomial analysis revealed that the pair of aflatoxin B1 and aflatoxin Q1 differed linearly and quadratically from the pair of aflatoxin B2 alpha and aflatoxicol, but within each pair there were no differences. Limited data with aflatoxin M1 revealed that it was slightly more active than aflatoxicol in the assay, but minimal replication prevented rigorous statistical testing. It may be theorized that the moderate to strong inhibition of blastogenesis by aflatoxin B1 and its metabolites could inhibit T lymphocyte functions, such as killer, helper, effector, or other immune processes, and thus compromise the immunological surveillance mechanism.

Metabolic effects of low aflatoxin B1 levels on broiler chicks.

The effects of daily ingestion of aflatoxin B1 (AFB1) on growth, feed intake, plasma glucose, plasma cholesterol, plasma amino acids, plasma albumin, plasma ceruloplasmin, muscle amino acids, liver lipid, and bone strength were studied. For 3 weeks, beginning at an age of 2 days, broiler chicks were dosed daily per os with 50 or 100 micrograms of AFB1 per kg of body weight. Body weight and feed consumption were recorded daily, and metabolic responses were determined at 3 weeks. Treatment with AFB1 did not significantly alter body weight or feed intake. Relative liver weight showed a significant increase at the highest dose, with a significant concomitant increase in liver lipid and decrease in hepatic zinc. Relative spleen and heart weights were not affected by the toxin. Plasma glucose and cholesterol were significantly elevated at the highest dose. AFB1 significantly decreased plasma lysine and histidine and significantly increased muscle histidine, arginine, and valine. AFB1 decreased plasma albumin and markedly increased plasma ceruloplasmin. Dimensions of the long bones (femur and tibiotarsus) were not altered by the toxin. However, AFB1 caused a significant linear decline in the resistance of bone to breakage ("bone breaking strength"). The results indicate that low levels of AFB1 reduced bone strength in broiler chicks. The alterations in blood parameters indicated that AFB1 can disrupt metabolism even at low levels.