Isolation, characterization and synthesis of alpha-foetoprotein from neonatal-rat skin.

Monospecific anti-[rat alpha-foetoprotein(alpha-FP)] immunoglobulin G was coupled to CNBr-activated Sepharose-4B (4.5 mg/ml packed volume of gel) to yield an adsorbent. The immunoaffinity column was used to isolate alpha-FP from neonatal-rat skin. Purified skin alpha-FP was found to be immunologically and electrophoretically similar to serum alpha-FP. It yielded a single band with mol.wt. 68000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. However, on polyacrylamide-gel electrophoresis under non-denaturing conditions, the alpha-FP displayed slow- and fast-moving variants similar to those observed in serum alpha-FP. A Scatchard plot of oestradiol binding to the alpha-FP yielded an association constant of 2.5 X 10(9)M-1 by dextran-coated-charcoal and 0.75 X 10(8)M-1 by Sephadex-gel-filtration procedures respectively. Skin explants from newborn rats were found to incorporate [14C]leucine into immunoprecipitable intracellular alpha-FP. Cycloheximide inhibited the synthesis of alpha-FP in skin explant culture. Our results indicate that newborn-rat skin contains alpha-FP that is similar to serum alpha-FP and which may arise in neonatal-rat skin as a result of synthesis in situ.     

Participation of the lone tryptophan residue of rat alpha-foetoprotein in its drug-binding sites. Comparison with rat serum albumin.

The participation in drug binding of the lone tryptophan residue of rat alpha-foetoprotein (alpha-FP) and serum albumin, the two main transport proteins of foetal serum, has been studied by two different techniques. Firstly, the effect on phenylbutazone and warfarin binding of the chemical derivatization of the lone tryptophan residue of both proteins by 2-nitrophenylsulphonyl chloride (NPS) was studied. Secondly, the effect of phenylbutazone binding on the intrinsic fluorescence of the tryptophan residue of rat alpha-FP and albumin was investigated. The specific modification of the proteins by NPS did not affect the binding of warfarin by rat alpha-FP and albumin, but greatly decreased the affinity of the high-affinity sites of rat alpha-FP for phenylbutazone, though the numbers of these sites were not significantly changed. However, for albumin a similar decrease in the affinity constant appeared to be due to the reaction conditions. The spectrofluorimetric studies showed that the lone tryptophan residue of alpha-FP and albumin was quenched by phenylbutazone binding, and the quenching paralleled the fractional saturation of the high-affinity site only in the case of albumin. The effect of phenylbutazone binding on the intrinsic fluorescence of rat alpha-FP indicated that the lone tryptophan residue of this foetal protein is not in the same molecular environment as that of albumin, not participating directly in the high-affinity site for phenylbutazone, and the effect may be via some induced conformational change in rat alpha-FP. These results also confirm our previous suggestion that the high-affinity sites for phenylbutazone and warfarin are different on the rat alpha-FP molecule. The results seem to indicate that this is also the case for albumin, but confirmation is necessary.     

Dynamics of alpha-fetoprotein concentration in mice of different genotypes during the neonatal period]

A comparative assay of the alpha-fetoprotein (alpha-FP) level in mice of various genotypes (CBA, C3H, C57BL/Se/Sn, BALB/c, CC57W, AKR and nude--nu/nu) was conducted in the course of 3 weeks of postnatal period. The concentration of alpha-FP reached the following levels: the first day 2(-10)-2(-9); the 5th day 2(-8); the 8th day 2(-7); the 15th day 2(-4); on the 22nd day the level was zero. Nude mice which showed the alpha-FP concentration of 2(-2) on the 15th day were an exception. A conclusion was drawn that the alpha-FP synthesis was based not on the athymia of nude mice per se, but upon other unknown factors.     

Influence of the culture time on DNA damage and repair in isolated rat hepatocytes exposed to nitrochlorobenzene derivatives

The induction of DNA damage on 1.5- and 24-h cultured hepatocytes was tested after a 3-h exposure to 5 and 50 microM mono-, di-, and trinitrochlorobenzene (100-00-5; 97-00-7; 88-88-0). DNA-repair synthesis, elicited by nitrochlorobenzene treatment, was also estimated 24 and 48 h after the withdrawal of the nitro-aryl halides. DNA damage and repair were evaluated by determining the DNA elution rate in alkali. A dose-related rate of DNA damage was obtained by exposure of 1.5-h-cultured hepatocytes to 5 and 50 microM nitrochlorobenzenes . DNA of 24-h-cultured cells was not affected by nitrochlorobenzene treatment. The data obtained by exposure to 5 microM methyl methanesulfonate (66-27-3) and nitrosodimethylamine (62-75-9), direct and indirect methylating agents, suggest that 24-h-cultured liver cells are still able to transform nitrosodimethylamine but not nitrochlorobenzenes . Isolated hepatocytes maintain their capability of repairing the induced DNA damage when cultured for 24 and 48 h in fresh medium. The system offers an interesting model to investigate the perturbations related to the metabolism of xenobiotics.     

Fetal proteinuria in diagnosis of congenital nephrosis detected by raised alpha-fetoprotein in maternal serum.

High concentrations of alpha-fetoprotein (alpha-FP) were found at 14, 19, and 21 weeks gestation in the serum of a woman with a history of unexplained fetal death in her previous pregnancies. The alpha-FP concentration of the liquor also was high at 21 weeks and the pregnancy was terminated. Though the fetus was macroscopically normal, measurement of albumin, alpha-FP, IgG, and alpha2-macroglobulin in the fetal urine showed a selective proteinuria, and congenital nephrosis was diagnosed after examination of the fetal kidneys by electron microscopy. Possibly some fetuses reported to be "false-positive for neural tube defect" may have had renal lesions of this nature. Examination of fetal urine may be the simplest initial diagnostic procedure in any future case.     

Reestablishment of the heterogeneous distribution of hepatic glutamine synthetase during regeneration after CCl4-intoxication

Intoxication of rats with CCl4 (1 ml/kg) resulted in the almost complete loss of glutamine synthetase (GS) specific activity and immunologically detectable enzyme protein known to be expressed exclusively in some hepatocytes of the perivenous zone of the liver acinus. During regeneration the specific activity as well as the original number of GS-positive (GS+) hepatocytes were reestablished. However, while the GS+ hepatocytes in control livers were arranged in up to 3 cell layers surrounding the central veins the same number of GS+ hepatocytes in regenerated livers formed a single cell layer only, most likely because the central veins were enlarged in diameter. Investigation of the nuclear pattern of GS+ and GS- hepatocytes of control animals in primary cultures revealed striking differences characterized by significantly more mononuclear diploid, binuclear diploid, and binuclear tetraploid cells among the GS+ hepatocytes and predominantly mononuclear tetraploid cells (70%) among the GS- hepatocytes. Immediately after liver damage by CCl4 and during regeneration small but significant changes in the nuclear pattern were noted for GS- hepatocytes. However, the first GS+ cells appearing during early regeneration showed a pattern of ploidy classes close to the original one found for GS- hepatocytes. These results indicate that new GS+ hepatocytes may be derived from formerly GS- cells which are induced to express GS if they have reached the border of the central veins.     

Changes in the rhythms of the alpha-fetoprotein content of the blood serum of mice with hepatoma 22A and of tumor cell proliferation depending on the time of cyclophosphane administration

A study was made of the content of alpha-fetoprotein (alpha-FP) in the blood serum and of proliferative processes in hepatoma 22A-bearing mice after injection of cyclophosphamide in a dose of 150 mg/kg at varying time of 4-day tumor growth. The data obtained indicate that the time of appearance of alpha-FP in the blood, the pattern of rhythmic fluctuations in protein content and in the number of DNA-synthesizing and mitotic cells of the tumor depend on the time of the cytostatic injection during the day. Besides, cyclophosphamide exerts a more powerful inhibitory effect on the content of alpha-FP in the blood serum and less powerful on the proliferative processes in hepatoma 22A.     

Separation of hepatocytes of different acinar zones by flow cytometry

Hepatocytes in the proximal (zone 1) and distal (zone 3) regions of the liver acinus are selectively stained by perfusion of the isolated rat liver with 0.2-20 microM acridine orange (AO). After 10-60 min of anterograde perfusion, AO fluorescence is visible in zone 1 cells, whereas retrograde perfusion stains cells of zone 3. In this paper, we describe a technique to isolate a mixed population of fluorescent and nonfluorescent hepatocytes (cells from all acinar zones, which do not loose the zone specific AO labeling) and to separate these cells according to their zonal origin by fluorescence activated cell sorting. The zonal populations obtained were either fluorescent or nonfluorescent (purity greater than 95%). Separated cell fractions differed in their enzyme content (5' nucleotidase, succinate-dehydrogenase, beta-glucuronidase). An unidentified AO metabolite, which is not found in bile after retrograde perfusion (not formed in zone 3 cells), is also absent after retrograde perfusion in sorted fluorescent cells (zone 3 cells), indicating zonal purity of sorted cells.     

Morphometric study of alterations of extrafocal hepatocytes of rat liver treated with N-nitrosomorpholine

Male Sprague-Dawley rats were treated with the hepatocarcinogen N-nitrosomorpholine for 7 weeks and observed for up to 40 weeks after withdrawal of the carcinogen. In addition to the focal preneoplastic lesions described earlier there were also alterations in extrafocal hepatocytes and these changes have been quantified morphometrically. Since the periportal and perivenous hepatocytes were not altered to the same extent, cells of the periportal zone (PPZ) and cells of the perivenous zone (PVZ) were measured separately. Immediately after stopping treatment there was marked enlargement of the hepatocellular cytoplasm and of the nuclei in both the PPZ and the PVZ and a reduction in the number of binuclear hepatocytes. While these alterations were totally reversed during the first 10 weeks after treatment ceased in the PPZ, statistically significant changes in nuclear size, nuclear-cytoplasmic ratio and number of binuclear cells persisted in the PVZ up to 40 weeks after the end of treatment. We suggest that both the foci of altered hepatocytes as well as the persisting changes in extrafocal hepatocytes may be involved in the process of hepatocarcinogenesis.     

Mouse alpha 1-fetoprotein and albumin. A comparison of their binding properties with estrogen and fatty acid ligands

The binding of estradiol-17 beta (E2), diethylstilbestrol (DES), and polyene fatty acids, in particular arachidonate (C20:4), to alpha 1-fetoprotein (alpha-FP) and albumin purified from mouse embryo sera was studied using equilibrium dialysis and electrophoretic techniques. E2, arachidonate, and DES all bind to alpha-FP, but with decreasing strength. E2 is a high affinity, low capacity ligand (Ka approximately 0.8 X 10(8) M-1 and approximately 0.3 sites/mol of alpha-FP at 25 degrees C); arachidonate is a weaker ligand disposing of more sites (Ka approximately 0.3 X 10(7) M-1 and 4-5 sites/mol of alpha-FP); the binding of DES is of comparatively low affinity and capacity (Ka approximately 0.2 X 10(7) M-1 and n approximately 0.7/mol of alpha-FP). In spite of different structures and equilibrium parameters, E2, DES, and arachidonate are able to compete with each other for binding to the fetoprotein. The C22:4 and C22:6 fatty acids are also efficient concentration-dependent inhibitors of E2 or DES binding. Albumin binds the fatty acids and DES, but equilibrium parameters are different from those of alpha-FP. In particular, arachidonate is a better ligand for albumin, where it interacts with at least two classes of apparent sites (Ka1 approximately 0.3 X 10(8) M-1 and n1 approximately 1; Ka2 approximately 0.2 X 10(7) M-1 and n2 approximately 30). In contrast to alpha-FP, albumin virtually does not bind E2. Also, no competition could be demonstrated between DES and fatty acid ligands for binding to albumin. None of the studied interactions, with either albumin or alpha-FP, was modified even by high doses of bilirubin. The possible functions of the various binding activities present in fetal sera in the process of growth are discussed.     

Radiation sensitivity of adult human parenchymal hepatocytes

The radiosensitivity of human hepatocytes was determined and compared to that of rat hepatocytes. This interspecies comparison was performed by using the alkaline elution technique to measure DNA single-strand breaks and their repair in irradiated primary cultures of hepatocytes. Human hepatocytes obtained from discarded surgical material and Fischer 344 female rat hepatocytes were enzymatically dispersed with collagenase, placed in culture, and irradiated with 0, 10, 20, and 40 Gy of 60Co gamma rays. The DNA was eluted either immediately after irradiation or at different times following incubation at 37 degrees C to allow for DNA single-strand break repair. The slopes of the dose-response relationship (strand scission factor versus dose) without DNA repair were 0.014 +/- 0.002 Gy-1 (n = 5) and 0.018 +/- 0.003 Gy-1 (n = 12) in human and rat hepatocytes, respectively; they were not significantly different. The half-time for fast and slow repair in human and rat hepatocytes was also not significantly different (i.e., 17.8 +/- 4.4 min and 253 +/- 67 min, and 13.9 +/- 6.1 min and 121 +/- 31 min, respectively), and 15 to 25% of the initial radiation-induced DNA damage was still present after 3 h of repair.     

Streptozotocin-induced liver damage in mice

Diabetes incidence and liver damage was studied and identified in C3H-s mice 21 days after Streptozotocin (SZ) administration (250 mg/kg/i.v.) at 04 hs (4 a.m.) and 16 hs (4 p.m.). Metabolic disturbances were assessed by daily control of glycosuria and serum glucose determined at the end of the experiment. Liver damage was controlled by light and electron microscopy. Both effects showed a circadian variation, with significant greatest values in the 16-h-injected group. Liver damage appeared whether the animals became diabetic or not, consisting in degranulation of the rough-surfaced endoplasmic reticulum, mitochondrial swelling with loss of cristae and edema of the ground substance, with flocculent amorphous precipitate. In some hepatocytes, a dilated cisternae of the endoplasmic reticulum was seen. It was concluded that: a) beta-cell and hepatocytes have a synchronic circadian sensitivity to SZ; b) liver damage was present whether the animals became diabetic or not, suggesting the presence of a different threshold for SZ effect in hepatocytes. These results might be taken into account when planning SZ use, either for experimental or clinical purposes.     

Changes in intra- and extrahepatic VLDL in the rat following acute injury by thioacetamide. A morphometric and biochemical study

The effect of a single dose of TAA (100 mg/kg body weight) on intra- and extrahepatic lipoproteins of the very low density (VLDL) type was studied in rats by morphometric and biochemical methods. For a better quantification of VLDL in the periportal (zone 1) and centrilobular (zone 3) hepatocytes of control and TAA-intoxicated livers, colchicine was used as an inhibitor of hepatic lipoprotein secretion. Generally, there exists a great functional heterogeneity between the hepatocytes of zone 1 and 3 in the colchicine-treated controls manifested in a significantly different accumulation rate of VLDL particles. The mean number of VLDL particles per vesicle, the mean secretory vesicle size and the volume density of the electron-lucent secretory vesicles are two times larger in hepatocytes of zone 1 than zone 3. The volume of the VLDL particles amounts to 7.3 X 10(-5) microns3 and 22 X 10(-5) microns3 in the peri- and centrilobular regions. On the other hand, there is no significant lobular-zonal difference in the number of light and dark secretory vesicles. Within 48 h TAA treatment causes a reduction in the number of VLDL particles/100 microns 2 in zone 1 and 3 by 66% and 61%, whereas the number of light secretory vesicles is decreased by 31% and 58%, respectively. The volume density of the latter is significantly diminished only in zone 1. Moreover, the VLDL particle volume is reduced to nearly 50% in each lobular zone examined. The data obtained after TAA treatment from the electron-dense secretory vesicles do not differ significantly from those of the colchicine-treated controls. Acute TAA intoxication lowers the hepatic VLDL-TG output by about 50% in comparison with controls. The steady state of the serum TG concentration after TAA application implies that the clearance of TG from the serum must be diminished to the same extent as the hepatic TG output is found to decrease due to acute liver injury. The results presented here support our view that acute TAA intoxication lowers the hepatic VLDL output by inhibiting the intracellular formation of VLDL. The intrahepatic degradation of the newly synthesized VLDL seems to be unaffected. Despite the fact that the substructure of the hepatocytes in zone 3 is much more changed than in zone 1 after TAA treatment the quantitative data on the VLDL secretory products provide evidence that the process of lipoprotein formation is disturbed to nearly the same extent by TAA both in zone 1 and 3.     

Functional heterogeneity of rat hepatocytes: predominance of aryl hydrocarbon hydroxylase activity in perivenular zone

To elucidate the hepatic intralobular distribution of aryl hydrocarbon hydroxylase (AHH) activity biochemically, periportal (PP) and perivenular hepatocytes (PV) from male Sprague-Dawley rats were separated by a fluorescence-activated cell sorter after labeling the PP zone with fluorescein diacetate and the perivenular zone with fluorescein isothiocyanate. AHH activity was higher in PV than in PP. The enzyme activity was induced about 6-fold in hepatocytes of rats pretreated with 3-methyl-cholanthrene, and the induction was more prominent in PP than in PV. Neither phenobarbital pretreatment nor altered lipid content of the diet induced the change in the enzyme activity.     

Tissue and subcellular distribution of glucokinase in rat liver and their changes during fasting-refeeding

The distribution of glucokinase in rat liver under both normal feeding and fasting-refeeding conditions was investigated immunohistochemically. Under normal feeding conditions, glucokinase immunoreactivity was observed in both nuclei and cytoplasm of parenchymal cells. The nuclei were stained intensely and evenly, whereas the cytoplasm showed weak immunoreactivity of different degrees of staining intensity depending on the location of the cells. The cytoplasm of perivenous hepatocytes was stained more intensely, though not so much more, than that of periportal hepatocytes. The cytoplasm of hepatocytes surrounding the terminal hepatic venule (THV), of hepatocytes surrounding the portal triad, and of some other hepatocytes showed a stronger immunoreactivity than that of residual hepatocytes. The nuclear immunoreactivity in hepatocytes surrounding the portal triad and in some other hepatocytes was weak or absent, and positive immunoreactivity was detected at the plasma membrane of some of these cells. After 72 h of fasting, glucokinase immunoreactivity was markedly decreased in all hepatocytes. After the start of refeeding, the cytoplasmic immunoreactivity began to increase first in the parenchymal cells surrounding the THV and extended to those in the intermediate zone followed by those in the periportal zone. In contrast, the increase in nuclear immunoreactivity started in hepatocytes situated in the intermediate zone adjacent to the perivenous zone and then extended to those in the perivenous zone followed by those in the periportal zone. Hepatocytes surrounding either THV or portal triad showed a distinctive change in immunoreactivity during the refeeding period. After 10 h of refeeding, strong immunoreactivity was observed in both the cytoplasm and the nuclei of all hepatocytes, and appreciable glucokinase immunoreactivity was detected at the plasma membrane of some hepatocytes. These findings are discussed from the standpoint of a functional role of glucokinase in hepatic glucose metabolism.     

Reestablishment of the heterogeneous distribution of hepatic glutamine synthetase during regeneration after CCl4-intoxication

Summary Intoxication of rats with CCl₄ (1 ml/kg) resulted in the almost complete loss of glutamine synthetase (GS) specific activity and immunologically detectable enzyme protein known to be expressed exclusively in some hepatocytes of the perivenous zone of the liver acinus. During regeneration the specific activity as well as the original number of GS-positive (GS⁺) hepatocytes were reestablished. However, while the GS⁺ hepatocytes in control livers were arranged in up to 3 cell layers surrounding the central veins the same number of GS⁺ hepatocytes in regenerated livers formed a single cell layer only, most likely because the central veins were enlarged in diameter. Investigation of the nuclear pattern of GS⁺ and GS^– hepatocytes of control animals in primary cultures revealed striking differences characterized by significantly more mononuclear diploid, binuclear diploid, and binuclear tetraploid cells among the GS⁺ hepatocytes and predominantly mononuclear tetraploid cells (70%) among the GS^– hepatocytes. Immediately after liver damage by CCl₄ and during regeneration small but significant changes in the nuclear pattern were noted for GS^– hepatocytes. However, the first GS⁺ cells appearing during early regeneration showed a pattern of ploidy classes close to the original one found for GS^– hepatocytes. These results indicate that new GS⁺ hepatocytes may be derived from formerly GS^– cells which are induced to express GS if they have reached the border of the central veins.     

Inhibitors of Protein Biosynthesis Can Stimulate Proliferation of Mouse Hepatocytes in Vitro

Hepatocyte proliferation in the liver regenerating after partial hepatectomy ceases when the organ is restored, and the mechanism of this phenomenon is still unclear. In the experiments on fusing hepatocytes from the regenerated mouse liver (15 days after partial hepatectomy) with NIH 3T3 mouse fibroblasts, we revealed no DNA synthesis in the nuclei of stimulated fibroblasts in heterokaryons (in the presence of hepatocyte nuclei), whereas DNA synthesis in nonfused cells was undisturbed. In this work, our purpose was to find out whether the suppression of DNA synthesis in heterokaryons could be due to the appearance in hepatocytes of some endogenous factors having an inhibitory effect on proliferation. To this end, hepatocytes from the mouse liver regenerated after partial hepatectomy were treated with cycloheximide for 1–4 h and were then fused with stimulated fibroblasts. Such a short-term treatment of hepatocytes with cycloheximide proved to result in the loss of their ability to inhibit DNA synthesis in the nuclei of stimulated or quiescent fibroblasts in heterokaryons, but hepatocytes proper actively proliferated in the medium with a low serum content (0.2%). When the mice with the liver regenerated after partial hepatectomy were treated with a single sublethal dose of cycloheximide (3 mg/kg), their hepatocytes taken two days after this treatment had no inhibitory effect. Puromycin, another inhibitor of protein synthesis, had the same effect on hepatocytes. These results may be interpreted as evidence that the final stage of liver regeneration after damage is controlled by the factors having a negative effect on cell proliferation.     

体外冲击波碎石术对兔肝酶活性的影响

采用超微组织化学方法,观察了体外冲击波碎石术(ESWL)对家免肝脏酶活性的影响。实施 ESWL 后,肝细胞的琥珀酸脱氢酶(SDH)和毛细胆管壁上的碱性磷酸酶(ALP)、焦磷酸硫胺素酶(TPPase)反应活性减弱或消失。TPPase 从损伤的肝细胞高尔基体分泌面扁囊、溶酶体样小泡和毛细胆管内溢出,并伴有肝细胞面的质膜上出现了 TPPase 反应产物和形成膜包内凹小泡。结果提示 ESWL 可对肝细胞及毛细胆管的功能和结构有损伤作用。     

The induction of chromosomal damage in rat hepatocytes and lymphocytes I. Time-dependent changes of the clastogenic effects of diethylnitrosamine,dimethylnitrosamine and ethyl methanesulfonate

Male Wistar rats received a single injection of diethylnitrosamine (DEN), dimethylnitrosamine (DMN) or ethyl methanesulfonate (EMS). After a number of time intervals (up to 56 days) liver cells were assayed for the presence of possible preclastogenic damage by performing partial hepatectomy and subsequent analysis of chromosomal damage (micronucleus formation) in isolated hepatocytes. Peripheral blood lymphocytes from the same animals were collected, stimulated to proliferate and assayed for the frequency of sister-chromatid exchanges (SCEs). Whereas all agents significantly increased frequencies of SCEs in lymphocytes up to at least 28 days (EMS) or 56 days (DMN, DEN) after injection, only the latter 2 compounds gave rise to significantly increased incidences of micronucleated hepatocytes. DMN-induced preclastogenic damage in hepatocytes was lost between 28 and 56 days after injection. After DEN, this type of damage was persistent over the entire experimental period (56 days).When rats treated with DEN did not undergo partial hepatectomy, the frequencies of micronuclei at different time intervals after treatment were at control level. This result, together with those from hepatectomized DEN-treated rats, suggests that it is the persistent character of the preclastogenic damage that is responsible for the occurrence of micronucleated hepatocytes at later time intervals after treatment with DEN, rather than the stability of micronuclei which might eventually have been formed soon after injection.