W. N. Holmes

The effects of ingested petroleum on the naphthalene-metabolizing properties of liver tissue in seawater-adapted mallard ducks (Anas platyrhynchos)

Abstract Hepatic mixed function oxidase activities were estimated in seawater-adapted mallard ducks ( Anas platyrhynchos ) that had been consuming food contaminated with one of five different types of crude oil. After 50 days of exposure to contaminated food, enzyme activities of liver microsomal preparations were assessed in terms of their naphthalenemetabolizing properties in vitro . Although dose-dependent increases in the total hepatic enzyme activities (nmole naphthalene metabolized per minute per unit mass body weight) were observed in birds consuming food contaminated with each type of crude oil, three patterns of response were apparent. Crude oils from South Louisiana and Kuwait stimulated large and significant increases in the specific activity of the enzyme system (nmole naphthalene metabolized per minute per unit mass microsomal protein), whereas little or no increase in either microsomal protein content or relative liver weight were observed. In contrast, two crude oils from Santa Barbara, Calif., induced only small increases in specific activity but significant increases occurred in hepatic microsomal protein concentration and relative liver weight. The crude oil from Prudhoe Bay, Ala., evoked intermediate patterns of response. The possible significance of these data is discussed in relation to the survival of seabirds consuming petroleum-contaminated food and drinking water.

Evidence for the zonation of interrenal tissue in the adrenal gland of the duck (Anas platyrhynchos)

SummaryAlthough interrenal tissue from the intact duck does not show a clear zonation when examined by light microscopy, the tissue does develop a well defined zonation following exposure to high and low levels of corticotropic stimulation. Under these conditions clear ultrastructural differences are seen between cells of the subcapsular zone (SCZ) and the inner zone (IZ). Based on these observations, the ultrastructure of tissue from intact birds was examined retrospectively and in addition, cell sizes and the relative volumes and areas of intracellular components were measured morphometrically. These analyses reveal morphological and quantitative differences between cells from the IZ and the SCZ. Cells of the IZ have small rounded nuclei, extensive smooth endoplasmic reticulum (SER) and mitochondria with tubular cristae, whereas cells from the SCZ contain pleomorphic nuclei, less SER and mitochondria with shelf-like cristae. The mean cell volume in the IZ is significantly less than that in the SCZ. In the SCZ cells the volume densities (volume per unit volume cytoplasm) of mitochondria and lipid droplets and the surface densities (area per unit volume cytoplasm) of the outer mitochondrial membranes are significantly greater than those in IZ cells. Conversely, in the cells of the IZ the volume densities of the nuclei and dense bodies and the surface density of the SER are greater than the corresponding values estimated for the cells of the SCZ. Although the mitochondria comprise a smaller fraction of the mean volume of IZ cells than SCZ cells, the total surface area of the cristae is approximately the same in the cells of both zones.

The effects of colchicine, vinblastine and cytochalasins on the corticotropic responsiveness and ultrastructure of inner zone adrenocortical tissue in the Pekin duck

SummaryTissue slices superfused with medium containing no ACTH released only traces of corticosterone. Addition of ACTH to the medium caused the rate of corticosterone release to increase to a maximum about 45 min after the addition of ACTH, after which time it either remained constant or started to wane slightly. The rate of release was affected by tissue thickness; the maximum rate of corticosterone release occurred when the tissue slices were 200 μm. Stimulated adrenocortical cells had large spherical nuclei, numerous mitochondria with tubular cristae, numerous lipid droplets, and a large amount of smooth endoplasmic reticulum. Many cells had an extensive network of microfilaments adjacent to the plasma membrane and some microtubules. Prolonged superfusion caused degenerative changes in some of the cells. Both cytochalasin B and cytochalasin D, dissolved in DMSO before addition to the superfusion medium, inhibited the corticotropic responsiveness in a dose-dependent manner. Control tissue samples superfused with medium containing DMSO, but no ACTH and no cytochalasin, released significantly more corticosterone than corresponding unstimulated samples. Few or no microfilaments were observed in adrenocortical cells after treatment with cytochalasin. Neither colchicine nor vinblastine had any discernible effect on the corticotropic responsiveness. After treatment with colchicine, adrenocortical cells had an ultrastructure characteristic of inner zone stimulated cells except that they were mainly devoid of microtubules.

The fine structure of the interrenal cells of the duck (Anas platyrhynchos) with evidence for the possible exocytotic release of steroids

SummaryThe duck interrenal cell possesses ultrastructural characteristics common to other steroid-secreting cells. Lipid droplets and mitochondria are abundant and lie principally at the apical end of the cell. Lipid droplets are not membrane-limited. Cisternae of smooth endoplasmic reticulum that are occasionally continuous with the less abundant rough endoplasmic reticulum are a prominent feature of the interrenal cell. Tubular profiles of rough endoplasmic reticulum often lie tangentially to mitochondria and ribosomes are either free, grouped in polyribosomal clusters, or bound to the endoplasmic reticulum. Mitochondria possess tubular cristae in the inner regions of the gland and frequently contain a paracrystalline array of small 10nm (o.d.) tubules and less frequently a hexagonal array of 40nm trilaminar rings. Other cytoplasmic components include dense bodies, residual bodies, microtubules, microfilaments and specialized single membrane-bound vesicles. Gap junctions, intermediate junctions and interdigitating processes constitute the main intercellular associations. No tight junctions were identified.The single membrane-bound vesicles which are occasionally filled with a low electron-dense, lipid-like material form septate-like “junctions” with the plasma membrane. The septa bridge an intracellular gap of 15–17 nm. The vesicles are usually located near the subendothelial space at the basal and basilateral regions of the cell. Occasionally, vesicles fuse with the plasma membrane. It is suggested that these vesicles represent morphological evidence for the exocytotic release of steroid hormones.

Adrenocortical function and hepatic naphthalene metabolism in mallard ducks (Anas platyrhynchos) consuming petroleum distillates

Abstract The reductions in plasma corticosterone concentration and the increases in hepatic naphthalene-metabolizing activity that occur when mallard ducks are acutely exposed to petroleum-contaminated food are each caused by distinct molecular-size classes of compounds in the whole crude oil. The decrease in plasma corticosterone concentration only occurred in birds given food contaminated with proportionate volumes of distillation fractions with boiling point ranges of X to 205°C and 205 to 399°C. Significant increases in total hepatic naphthalene-metabolizing activity, however, were only induced when birds were given food containing distillation fractions with boiling point ranges of 205 to 399°C and 399 to 482°C. The residual material containing compounds with boiling points in excess of 482°C had no effect on either plasma corticosterone concentration or hepatic naphthalene metabolism. Although the combined effects of the two distillation fractions that affected plasma corticosterone were approximately equal to that of the whole crude oil, the combined effects of the fractions that induced increases in hepatic naphthalene metabolism were almost twice that of the crude oil. Also, the relative abundances of the four distillation fractions were not the same and differences between their toxic potencies were identified with respect to both physiological parameters.

Structural changes occurring in interrenal tissue of the duck (Anas platyrhynchos) following adenohypophysectomy and treatment in vivo and in vitro with corticotropin

SummaryAdrenal glands from ACTH-treated intact ducks and chronically adenohypophysectomized ducks showed clear zonation into a subcapsular zone (SCZ) and an inner zone (IZ). Adenohypophysectomy caused ultrastructural changes in the IZ but not in the SCZ cells. These included increases in lipid droplets, changes in mitochondrial cristae from tubular to shelf-like, and changes in the shape of the nuclei from spherical to crenated. These changes were reversed by treatment with ACTH. Also, cells of the IZ, but not the SCZ, of adrenals from intact birds given ACTH showed more SER, more dense bodies, fewer lipid droplets and more prominent Golgi complexes.IZ cells incubated in buffer containing no ACTH developed mitochondria with shelf-like cristae and numerous opaque granules in the matrix. Exposure to buffer containing ACTH caused the mitochondrial cristae to become tubular and the matrix granules either decreased in number or disappeared. The granules could be extracted by incubating sections with chelating agents. The mitochondria in SCZ cells did not respond structurally to the presence of ACTH in the incubation medium but the matrix granules, like those in IZ cells, responded to the presence of chelating agents.

Functional significance of interrenal cell zonation in the adrenal gland of the duck (Anas platyrhynchos)

SummarySlices of whole adrenal gland tissue, incubated in vitro in the presence of ACTH for 1 h and 2 h produced corticosterone and aldosterone in constant ratio (16∶1). Tangential slices taken from the region immediately below the connective tissue capsule and slices taken from deeper regions of the gland consisted primarily of cells conforming to the distinct structural characteristics of the subcapsular zone (SCZ) and inner zone (IZ) tissues respectively. When samples were incubated in the presence of ACTH for 1 h and 2 h, the interrenal cells of the SCZ produced relatively more aldosterone than cells taken from the IZ of the gland. The corticosterone: aldosterone ratio for the IZ after 1 h (68∶1) and after 2 h (102∶1) were ten times greater than the ratios for the SCZ after 1 h (7∶1) and after 2 h (10∶1). The SCZ slices were not more than 60 cells thick and consisted of cells arranged in cords. These cells contained irregular nuclei, mitochondria with shelf-like cristae and a moderate abundance of smooth endoplasmic reticulum. In contrast, the production of large amounts of corticosterone by the cells of the IZ was associated with tissue containing more vascular space than the SCZ and the cells contained large round nuclei surrounded by an abundance of smooth endoplasmic reticulum and the mitochondria had tubular rather than shelf-like cristae.

Changes in corticotropic responsiveness and mitochondrial ultrastructure of adrenocortical cells from the inner zone of the duck (Anas platyrhynchos) adrenal gland: The effects of cycloheximide, puromycin, and chloramphenicol

SummaryTissue slices from the inner zone of the adrenal showed no ultrastructural abnormalities after being superfused with Krebs-Henseleit buffer for more than 2 h. Slices superfused with medium containing no ACTH produced only traces of corticosterone and most of the mitochondria had shelf-like cristae. Exposure to medium containing ACTH (1 μg 1-24 ACTH · ml-1) caused a significant increase in the release of corticosterone and the mitochondria developed tubular cristae. Cycloheximide and puromycin inhibited, in a dose-dependent manner, the release of corticosterone from tissue slices superfused with medium containing ACTH. A low concentration of cycloheximide in the medium (0.02 μg · ml-1) had only a slight effect on hormone release but many of the mitochondria developed dark matrixes and shelf-like or disrupted cristae. At higher concentrations of cycloheximide (0.2 μg · ml-1) the release of corticosterone was suppressed and when the concentration was increased to 2.0 μg · ml-1 it was blocked completely; each of these higher concentrations of cycloheximide caused the mitochondria to develop dense matrixes and shelf-like cristae. The inhibitory effects of cycloheximide on the corticotropic responsiveness of superfused tissue slices were reversible. Dependent upon the concentration, the addition of puromycin to the medium also caused, either partial (1.0 and 10.0 μm · ml-1) or complete (100 μg · ml-1), suppression of hormone release from slices superfused with medium containing ACTH; at the same time, the formation of tubular cristae in the mitochondria was blocked. Chloramphenicol had no effect on either hormone release or mitochondrial ultrastructure in the adrenocortical cells from the inner zone of the gland. It is suggested that a specific protein, synthesized in the cytoplasm, is necessary to accomplish the conformational changes in the mitochondrial cristae that accompany the full elaboration of corticotropin-induced hormone synthesis.

Functional differences between two structurally distinct types of steroidogenic cell in the avian adrenal gland

SummaryThere are two regions of steroidogenic cells in the duck adrenal gland. An outer, subcapsular zone (SCZ), consisting of cells with irregularly shaped nuclei, shows relatively little smooth endoplasmic reticulum and mitochondria with shelf-like cristae. This region surrounds the inner zone (IZ) of the gland which is comprised of smaller cells with rounded nuclei, a more abundant smooth endoplasmic reticulum and mitochondria with tubular cristae. When samples of tissue from these distinct regions of the gland are superfused in vitro with media containing concentrations of 1–24 ACTH ranging from 100 to 1000 ng per ml (0.034 to 0.34 μM) the steroidogenic cells in both zones release corticosterone in a dose-dependent manner. The dose-responsiveness of both the SCZ and the IZ cells over this range is a complex quadratic function of the 1–24 ACTH concentration in the medium and the semilogarithmic linear portions of the dose-response curves are restricted to a narrow midrange of ACTH concentrations. Throughout the dose-response range, however, the steroidogenic cells of the IZ are more responsive to corticotropic stimulation than are the cells of the SCZ. The cells of the two zones are further distinguished by their responses to a challenge for a second time with medium containing 1–24 ACTH; the responses of the IZ cells to a second challenge were greater than those of the SCZ cells, and at a high concentration of ACTH the SCZ slices showed no significant second response.