Immunochemical characterization of developmental changes in rat hepatic hydroxysteroid sulfotransferase.

A major isoenzyme of hepatic androsterone-sulfating sulfotransferase (AD-ST) was purified from adult female rats. The activity was purified 122-fold over that found in the cytosol and showed a single protein band with a subunit molecular mass of 30 kDa after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme exhibited four isoelectric variants of subunits on denaturing isoelectrofocusing gels (pI = 5.8, 6.1, 6.7 and 7.2). Rabbit antiserum raised against the enzyme specifically detected AD-ST polypeptide in rat liver cytosol. Immunoblot analysis of liver cytosol from female and male rats at various ages showed good correlation between the levels of AD-ST activity and AD-ST polypeptide. Significant levels of AD-ST activity and polypeptide were detected in senescent male rats, though normal adult male rats have very low levels of AD-ST activity and protein. The relative content of the isoelectric variants of AD-ST were different in liver cytosol of weanling and adult females, indicating that age- and gender-related alterations of hepatic AD-ST activity are primarily determined by the levels of AD-ST polypeptide and the relative amounts of the four isoelectric variants of the enzyme.     

Subcellular localization of aldolase B

The localization of the aldolase B isozyme was determined immunohistochemically in rat kidney and liver using a polyclonal antibody. Aldolase B was preferentially localized in a nuclear region of hepatocytes from the periportal region and was absent in those from the perivenous region. Aldolase B was also preferentially localized in the proximal tubules and was absent in other structures of the renal cortex as well as in the renal medulla. Using reflection confocal microscopy, the enzyme was preferentially localized in a nuclear position in liver and renal cells, which was similar to the cellular and intracellular location found for the gluconeogenic enzyme fructose-1,6-bisphosphatase (Sáez et al. [1996] J. Cell. Biochem. 63:453-462). Subcellular fractionation studies followed by enzyme activity assays revealed that aldolase activity was associated with subcellular particulate structures. Overall, the data suggest that different aldolase isoenzymes are needed in the glycolytic and gluconeogenic pathways.     

Metabolism of the human serum beta-hexosaminidase isoenzymes in the rat: an in vivo and in vitro study

Plasma clearance of purified human serum beta-hexosaminidase isoenzymes was studied in the rat. The serum beta-hexosaminidase isoenzymes (A, B and P) showed a slow clearance from circulation compared to their tissue counterparts. After desialylation, the clearance rate of all serum isoenzymes was markedly enhanced. The uptake of native as well as desialylated serum beta-hexosaminidase isoenzymes was studied in rat liver nonparenchymal cells and hepatocytes. No detectable uptake of any native serum isoenzyme was noticed in either cell type. However, when these isoenzymes were desialylated by neuraminidase treatment, isoenzymes A and B were taken up by the nonparenchymal cells. No uptake was observed for the P form. None of the desialylated serum forms was taken up by hepatocytes.     

Age- and sex-related differences in sensitivity to hepatotoxic action of estragole in mice

As determined by blood activity of alanine- and aspartate aminotransferases, intraperitoneal injection of estragole in subcarcinogenic dose 300 mg/kg does not damage the liver of suckling off-spring of both sexes and of adult SWR/J males but drastically damages it in mature females of this strain. Castration only slightly decreases the resistance of males to hepatotoxic action of estragole but significantly increases it in females; exogenous administration of estradiole benzoate to castrated males decreases their resistance to the hepatotoxin, whereas administration of testosterone propionate to ovariectomyzed females does not elevate it. Morphologically, estragole damages the same number of liver cells in females and males, but in males it induces mostly hydropic degenerative, and in females--necrotic changes of hepatocytes.     

Immunohistochemical localization of albumin and in situ hybridization of albumin mRNA

Hepatocytes actively involved in albumin synthesis were identified by immunohistochemical method. In sections of perioidate-lysine-2 per cent (w/v) paraformaldehyde fixed normal rat liver, albumin was detected in all hepatocytes. At the ultrastructural level, albumin was localized in the rough endoplasmic reticulum and in Golgi complexes located near the nucleus in only a small subpopulation of hepatocytes, while all other hepatocytes contained albumin only in Golgi complexes located near the bile canaliculi. Stimulation of albumin synthesis by puromycin aminonucleoside-induced nephrosis resulted in an altered intracellular distribution of albumin at the light microscopic level. When examined at the ultrastructural level, albumin was localized in the rough endoplasmic reticulum as well as in Golgi complexes located near the nucleus in nearly all these hepatocytes. Hepatocytes with the potential to synthesize albumin were identified by in situ hybridization of albumin mRNA. In sections of 0.1 per cent (v/v) glutaraldehyde perfusion fixed normal rat liver, albumin mRNA was detected in the cytoplasm of only a few hepatocytes scattered throughout the lobule. Following stimulation of albumin synthesis by the induction of nephrosis, albumin mRNA was detected in the cytoplasm of the hepatocytes. The source of albumin in those hepatocytes which lacked albumin mRNA was identified in analbuminemic rats injected with rat albumin. At 6 h post injection, the light microscopic distribution of albumin in the liver of these animals was virtually indistinguishable from that in normal rat liver. At the ultrastructural level, injected albumin was localized in lysosomes and in Golgi complexes located near the bile canaliculi.     

Expression of 8-OHdG in Zosterisessor ophiocephalus from the Venetian lagoon,Italy

The aim of the present work was to evaluate the expression of 8-OHdG (8-hydroxy-2''-deoxyguanosine) in the benthic fish Zosterisessor ophiocephalus collected in two differently polluted sites of the Venetian lagoon (Porto Marghera and Caroman). We compared our data on 8-OHdG with those of CYP1A (Cytochrome P450, family 1, subfamily A, polypeptide 1), which is a well known biomarker for detoxification of contaminants. Immunohistochemistry with an antibody to 8-OHdG showed immunopositivity in nuclei of hepatocytes as well as in melanomacrophage centres of spleen and kidney, whereas an anti-CYP1A antibody exhibited positive immunostaining in the liver, kidney and ovary. The liver of males showed higher expression of both proteins than females. In animals from Porto Marghera site, the enzymatic assay for 8-OHdG exhibited higher levels in liver of males than in females. Western Blot analysis using the antibody anti-CYP1A recognized the presence of a band of about 60 kDa in the liver of males and females. Males exhibited a strong band, whereas in females the band showed a lower intensity. By using Real-Time PCR, the mRNA expression of CYP1A did not show any differences between males and females from each site, but it was at borderline significance level. Comparing the two sites, mRNA expression of CYP1A was significantly higher in the liver of both males and females from Porto Marghera than that of Caroman. The present data suggest that pollutants are bio-available as demonstrated by our biomarker analyses and may have a harmful effect on aquatic organisms such as Z. ophiocephalus. We report that the highest levels of hepatic 8-OHdG and CYP1A expression were detected in males, showing clear gender specificity.Key words: OHdG, CYP1A, fish, immunohistochemistry, molecular biology.     

Stability of hexokinases A, B and C and N-acetylglucosamine kinase in liver cells isolated from rats submitted to diabetes and several dietary conditions.

The effect of dietary and hormonal variations on the specific activities of hexokinase isoenzymes, N-acetylglucosamine kinase and pyruvate kinase isoenzymes in parenchymal and non-parenchymal liver cells was studied. Hexokinase D was markedly decreased in hepatocytes from animals fasted or fed on the carbohydrate-free diet as well as from diabetic rats, attaining a constant low level of about 17% of normal values. Pyruvate kinase L was also diminished in hepatocytes under the same experimental conditions. In contrast, the three high-affinity hexokinase isoenzymes A, B and C remained without variation in total amount or in their relative proportions in hepatocytes and non-parenchymal liver cells isolated from animals under the various conditions studied. N-Acetylglucosamine kinase activities also did not change either in parenchymal or in non-parenchymal liver cells under all conditions. The results are discussed in relation to the significance of N-acetylglucosamine kinase and the various hexokinase isoenzymes for the phosphorylation of glucose after dietary and hormonal manipulations.     

Morphology of the green livers in upstream migrants of Petromyzon marinus L.

A morphological comparison was made of the green livers of male and female lampreys (Petromyzon marinus L.) collected during the upstream (prespawning) migration. Light and electron microscope histochemistry for iron, and both thin sections and freeze-fracture replicas in the electron microscope, revealed some sexual dimorphism in these livers. Ferric iron is much more abundant in the liver of females and is present in the cytoplasmic matrix, in dense bodies, and in vacuoles of hepatocytes. The numerous vacuoles of females may be the deposition site of biliverdin and other bile components that would account for the darker green coloration of the liver compared to males. Hepatocytes in females are also characterized by prominent rough endoplasmic reticulum and Golgi apparatus that reflect the involvement of the cells in vitellogenesis. The presence of numerous lipid droplets in the hepatocytes of males indicates that the liver is an important storage site for fat. The lipid droplets are associated with electron-dense deposits of unknown nature. Large gap junctions typify the parenchymal cells of both male and female livers. Perisinusoidal and sinusoidal cells are similar to those in the nonparenchymal region in other vertebrate livers, namely, endothelial and Kupffer cells, lipocytes (Ito), and some granulated cells. The relationship of lipocytes to fibrous tissue and fibrogenesis is discussed.     

Bile salt sulfotransferase: alterations during maturation and non-inducibility during substrate ingestion

Development of the capacity for hepatic biotransformation of potentially toxic, endogenous compounds such as lithocholate may be dependent on induction by substrate or hormonal modulation. Our aim was to observe the ontogeny of hepatic sulfotransferase (ST) activity, a presumed detoxification pathway, and to determine the effect of substrate ingestion and cortisone administration on ST activity. Pregnant rats were fed a standard chow diet containing lithocholate; the maternal diet was continued during the suckling and weaning phase of the pups. Liver cytosol and serum were obtained from dams and from pups at weekly intervals from fetal life through 4 weeks of age. In controls, there was a progressive increase in hepatic ST activity from 6.2 +/- 2.9 pmol/mg protein per min, (mean +/- SEM) in fetal liver, 18.1 +/- 3.9 at 1 week, an 33.6 +/- 7.2 at 2 weeks to a peak of 56.4 +/- 11.8 at 3 weeks of age. In female rats older than 4 weeks of age, ST activity in hepatic cytosol was threefold higher than in males. There was a decline to adult levels (9.2 +/- 2.4 in males and 39.4 +/- 4.3 in females) at 56 days of life. Cortisone acetate administration had no effect on enzyme activity in pups except those 3 weeks old or older in which there was a precocious decrease in enzyme activity to adult levels. The administration of lithocholate caused a dose-related postnatal alteration of intrahepatic bile ducts manifest as cholangitis with ductular proliferation; hepatocytes were spared.(ABSTRACT TRUNCATED AT 250 WORDS)     

All hexokinase isoenzymes coexist in rat hepatocytes.

The cellular distribution of hexokinase isoenzymes, N-acetylglucosamine Kinase and pyruvate kinases in rat liver was studied. Hepatocytes and non-parenchymal cells with high viability and almost no cross-contamination were obtained by perfusion in situ of the liver with collagenase, with the use of an enriched cell-culture medium in all steps of cell isolation. Separation of hexokinase isoenzymes was done by DEAE-cellulose chromatography, and enzyme activities were measured by a specific radioassay. Cytosol from isolated hepatocytes contained high-affinity hexokinases A, B and C, in addition to hexokinase D. The last-mentioned represented about 95% of total glucose-phosphorylating activity. Only hexokinase A was found associated t the particulate fraction. Isolated non-parenchymal cells contained only hexokinases A, B and C. N-Acetylglucosamine kinase was measured with a specific radioassay and was found as a single enzyme form in both hepatocytes and non-parenchymal cells, with higher activities in the former. Pyruvate kinase isoenzyme L was present only in the hepatocytes and isoenzyme K only in the non-parenchymal liver cells, confirming that they are good cellular markers.     

Sex-specific constitutive expression of the pre-neoplastic marker glutathione S-transferase, YfYf, in mouse liver.

Hepatic glutathione S-transferase isoenzyme content has been investigated in both sexes of three inbred strains of mice (DBA/2, C3H/He, C57BL6). A polypeptide (Mr 24,800), which is immunologically related to Yf purified from rat lung, was found to be expressed as a major form in all male mouse livers but represented only a minor enzyme form in female mouse liver. Glutathione S-transferases comprising subunits with molecular masses of 25,800 (Ya) or 26,400 (Yb) were present in males and females of the three strains under investigation. Cytosolic isoenzymes from all strains and sexes were purified to apparent homogeneity and no significant inter-strain differences in the properties of the individual forms were observed. In addition, no differences were detected in the microsomal glutathione S-transferase content of the different strains or sexes.     

Sex- and gonad-affecting scent compounds and 3 male pheromones in the rat

This study was aimed at identifying sex pheromones of the rat (Rattus norvegicus). We characterized the volatiles and semivolatiles of rat preputial gland and voided urine by using gas chromatography-mass spectrometry (GC-MS) and quantified them by their GC areas (abundances) and percentage of GC areas (relative abundances). Although all the compounds other than 4-heptanone and phenol detected were shared by males and females, the quantities for some of these sex-common compounds exhibited sexual dimorphism and decreased with gonadectomy. Thus, these compounds might be sex pheromones. Among them, squalene from preputial glands and 2-heptanone and 4-ethyl phenol from urine were 3 major compounds. They were richer in males and could be suppressed by castration. Adding any of the 3 compounds (at a concentration higher than its physiological level in male urine) to castrated male urine (CMU) increased the attractiveness of CMU to sex-naive females. Adding the 3 together (at the levels in normal male urine) to CMU significantly increased the attractiveness of CMU to females. However, such combination did not fully restore females' preference for urine from intact males, suggesting that some other trace compounds such as 4-heptanone and phenol might also play some roles in sex attractiveness. Thus, squalene, 2-heptanone, and 4-ethyl phenol were indeed male pheromone molecules in rats. Our study also indicates that E,E-beta-farnesene and E-alpha-farnesene, both richer in females than males, might be putative female pheromones.     

Effects Of A Post-Weaning Cafeteria Diet In Young Rats: Metabolic Syndrome,Reduced Activity And Low Anxiety-Like Behaviour

Among adolescents, overweight, obesity and metabolic syndrome are rapidly increasing in recent years as a consequence of unhealthy palatable diets. Animal models of diet-induced obesity have been developed, but little is known about the behavioural patterns produced by the consumption of such diets. The aim of the present study was to determine the behavioural and biochemical effects of a cafeteria diet fed to juvenile male and female rats, as well as to evaluate the possible recovery from these effects by administering standard feeding during the last week of the study. Two groups of male and female rats were fed with either a standard chow diet (ST) or a cafeteria (CAF) diet from weaning and for 8 weeks. A third group of males (CAF withdrawal) was fed with the CAF diet for 7 weeks and the ST in the 8th week. Both males and females developed metabolic syndrome as a consequence of the CAF feeding, showing overweight, higher adiposity and liver weight, increased plasma levels of glucose, insulin and triglycerides, as well as insulin resistance, in comparison with their respective controls. The CAF diet reduced motor activity in all behavioural tests, enhanced exploration, reduced anxiety-like behaviour and increased social interaction; this last effect was more pronounced in females than in males. When compared to animals only fed with a CAF diet, CAF withdrawal increased anxiety in the open field, slightly decreased body weight, and completely recovered the liver weight, insulin sensitivity and the standard levels of glucose, insulin and triglycerides in plasma. In conclusion, a CAF diet fed to young animals for 8 weeks induced obesity and metabolic syndrome, and produced robust behavioural changes in young adult rats, whereas CAF withdrawal in the last week modestly increased anxiety, reversed the metabolic alterations and partially reduced overweight.     

Immunohistochemical localization of pyruvate kinase isoenzymes in chicken tissues.

A method for the localization of pyruvate kinase isoenzymes type L, M2 and M1 in tissue sections is described. Mono-specific antibodies directed against isoenzymes of pyruvate kinase from chicken and the peroxidase antiperoxidase method were used. The following preferential localizations of the isoenzymes in chicken tissues were observed: Pyruvate kinase M1 was found in skeletal muscle. The white muscle fibers were more intensely stained than the red. Some dark muscles (e.g., anterior latissimus dorsi) and the heart muscle showed no reaction with antiserum against pyruvate kinase M1. Pyruvate kinase type L was found in the hepatocytes and in kidney cortex. Pyruvate kinase type M2 was seen in the distal tubules of kidney, in hepatocytes and sinusoidal cells in liver, in lung, adipose tissue, and in the spleen mainly in the bursa dependent areas. Pyruvate kinase type M2 was detected in high concentrations in the granulation tissue of regenerating liver after partial hepatectomy. Liver sections of a hen bearing a pancreatic tumor showed an unusually high content of pyruvate kinase type M2 in some hepatocytes, which were each clustered to spots in the liver parenchyma. Thus, contrary to previous reports, the tissue distribution of isoenzymes in chicken is similar to that of other vertebrates.     

The effect of separate and consecutive administration of cytochrome P-450 inducers to rats on the localization of various isoforms of the enzyme in liver lobe cells

By means of immunohistochemical methods a study of the liver intralobular localization of cytochromes P-450b(+e) and P-450c(+d) has been carried out after separate and consecutive treatment of rats with phenobarbital (PB) and 3-methylcholanthrene (MC). PB-treatment leads to localization of P-450b in the centrilobular region, whereas homogeneous distribution of P-450c in the lobule is observed after MC-treatment. The consecutive treatment with PB and MC is accompanied by the localization of P-450c only in cells of the periportal region of the lobule. PB-treatment of rats after preliminary MC-injection also results in the periportal localization of P-450c, and a small quantity of P-450b is localized in hepatocytes of the centrilobular region. Hence, the consecutive treatment with inducers of different molecular forms of cytochrome P-450 is accompanied by the redistribution of these isoenzymes in parenchymatous cells of the liver lobule. That is confirmed also by biochemical testing and immunochemical analysis of the microsomal fraction of hepatocytes.     

Male and female meiosis in grasshoppers

Male meiosis in the grasshopper Stethophyma grossum is well known as an example of proximal chiasma localisation. An investigation of female meiosis in oocytes of this species shows that both the frequency and distribution of chiasmata are quite different from the male situation. Mean chiasma frequency per cell (14.98) in considerably higher in females than in males (11.28) which agrees with the trend established in other comparative studies of male and female meiosis. More strikingly, males and females also show not only different but quite opposite patterns of chiasma distribution. In spermatocytes of males, chiasmata are strictly localised proximally in most bivalents, but in oocytes of females very few chiasmata form in proximal regions and nearly all chiasmata form either in distal or interstitial regions. The genetical significance of these findings is considered.     

Alkaline phosphatase isoenzymes in human kidney and urine,immunohistochemical, immunological and biochemical characterization

Summary Human kidney contains two antigenetically distinct isoenzymes of alkaline phosphatase (AP): a liver type and an intestinal type. The intestinal type AP is a minor component (1%–4%) of the total AP activity: it is found only in the cytoplasm. Both isoenzymes are located, found by an immunohistochemical technique, in the proximal convoluted tubules. This histochemical result eliminates the possibility that the low intestinal AP content in the kidney might only originate from blood vessels, where the intestinal isoenzyme was also found. The renal isoenzymes contribute to urinary AP. Intestinal type AP in urine of healthy persons, 10%–40% of the total AP activity, was found after high speed centrifugation predominantly in the supernatant (100,000 g), the liver type mainly in the sediment. Biochemical characterization revealed that intestinal type AP in kidney and urine are identical and differ from the isoenzyme of intestinal mucosa only slightly in their electrophoretic mobility.     

Immunochemical detection of different isoenzymes of cytochrome P-450 induced in chick hepatocyte cultures.

This study investigated whether the same cytochrome P-450 (P-450) isoenzymes were inducible in cultures of chick-embryo hepatocytes as in the liver of chicken embryos. We purified two isoenzymes of cytochrome P-450 from the livers of 17-day-old-chick embryos: one of molecular mass approx. 50 kDa induced in vivo by the phenobarbital-like inducer glutethimide, and the second of approx. 57 kDa induced by 3-methylcholanthrene. Rabbit antiserum against the 50 kDa protein inhibited benzphetamine demethylase activity in hepatic microsomes (microsomal fractions) from glutethimide-treated chick embryo. Antiserum to the 57 kDa protein inhibited ethoxyresorufin de-ethylase activity in hepatic microsomes from methylcholanthrene-treated chick embryo. Cultured chick hepatocytes were treated with chemicals known to induce isoenzymes of P-450 in rodent liver. The induced P-450s were quantified spectrophotometrically and characterized by immunoblotting and enzyme assays. From these studies, chemical inducers were classified into three groups: (i) chemicals that induced a P-450 isoenzyme of 50 kDa and increased benzphetamine demethylase activity: glutethimide, phenobarbital, metyrapone, mephenytoin, ethanol, isopentanol, isobutanol, lindane, lysodren; (ii) chemicals that induced a P-450 isoenzyme of 57 kDa and increased ethoxyresorufin de-ethylase activity: 3-methylcholanthrene and 3,3',4,4'-tetrachlorobiphenyl; and (iii) the mono-alpha-substituted 2,3',4,4',5-pentabromobiphenyl, which induced both proteins and both activities. The immunochemical data showed that chick-embryo hepatocytes in culture retain the inducibility of glutethimide- and methylcholanthrene-induced isoenzymes of P-450 that are inducible in the liver of the chicken embryo.     

Factors influencing aggression toward females by male rats exposed to anabolic androgenic steroids during puberty

Previous results showed that male rats pubertally exposed to anabolic androgenic steroids (AAS) displayed aggression towards females in response to physical provocation. This experiment examined two factors that may modulate AAS-induced behavior towards females: olfactory cues and frustration. Gonadally intact males began one of three AAS treatments at puberty (D40): testosterone propionate (T), stanozolol (S), T+S, or vehicle control. To test for the relevance of olfactory cues in the elicitation of behavior toward females, a hidden neighbor paradigm was used. The proximal stimulus was an ovariectomized (OVX) female, estrogen plus progesterone (E+P) female, or an E+P female with tape-obstructed vagina (OBS). Distal olfactory cues from a hidden neighbor were delivered from a separate cage connected to the testing arena. The vaginally obstructed, sexually receptive female (OBS) was used to determine the effects of frustration on behavior by AAS males. Both sexual and aggressive behaviors were measured. The presence of distal olfactory cues had no effect on either sexual or aggressive behavior. In the presence of E+P and OBS females, all males displayed sex behaviors, not aggression. However, AAS males displayed significantly more aggression towards proximal OVX females than controls. AAS males mounted OBS females significantly more than controls, indicating a persistence of once rewarded behavior. These results suggest (1) proximal cues of the conspecific female are more salient than distal olfactory cues in determining behavior and (2) AAS males display frustration-induced persistence in response to vaginally obstructed receptive females.