Detection of gamma glutamyl transpeptidase in the livers of germfree and conventional rats

Germfree and conventional rats, 1-41 months of age, were examined for gamma glutamyl transpeptidase in frozen sections prepared from their livers. This histochemical marker has been associated with early changes which develop following exposure to known carcinogenic agents. Positive gamma glutamyl transpeptidase markers were detected in 86% of germfree rats over 4 months of age, and the gamma glutamyl transpeptidase foci were larger and more numerous as the animals aged, and the foci were prominent in rats with hepatic tumors. Gamma glutamyl transpeptidase foci were observed in 96% of livers from conventional rats at 11-20 months of age. The agents responsible for induction of the gamma glutamyl transpeptidase foci in the liver were not identified.     

Histochemical localization of gamma glutamyl transpeptidase in the paranodal region of peripheral nerve

Gamma glutamyl transpeptidase (GGT), an amino acid transport enzyme, was investigated in normal and degenerated sciatic nerve of rat. The enzyme activity, which is considered to be a marker for cerebrovascular endothelium, was found to be absent in microvessels of normal and degenerated nerves. In the perineurium of normal nerve, GGT activity was faint, while in degenerated nerve, it increased. The most striking finding of this study was the observation of GGT activity at the paranode of each normal myelinated axon. It is interesting that after axotomy (8 weeks), no GGT activity was observed in the Schwann cells of degenerated nerve. Thus, Schwann cell plasmalemma contributed to GGT staining only when this cell was in contact with an axon mature enough to cause it to produce myelin. We conclude that, in peripheral nerve, transmembrane amino acid transport is apparently regional and associated with the paranodal region of myelinated nerve fibers.     

二乙基亚硝胺诱导大鼠肝癌模型的建立及评价

目的:通过间断小剂量二乙基亚硝胺(DEN)腹腔注射的方法,建立大鼠肝癌模型,并进行评价.方法:60只健康雄性SD大鼠随机分为C组(对照组,n=10)和M组(造模组,n=50),其中M组动物给予DEN腹腔注射,按体重20mg/kg给药,每周3次,至12周时停药.C组给予同等剂量和频率的生理盐水腹腔注射.观察两组动物不同时期肝脏大体变化及病理改变.结果:16周时诱癌成功率为76.2%(32/42).病理学结果显示,M组动物肝脏病变过程经历肝细胞损伤坏死期、肝细胞增生硬化期和肝细胞癌变期.结论:通过间断小剂量DEN腹腔注射的方法,成功建立了大鼠肝癌模型,该模型可作为一种理想的研究人类肝癌发生的动物模型.     

Rho-kinase inhibitor prevents hepatocyte damage in acute liver injury induced by carbon tetrachloride in rats

A protective effect of Rho-kinase inhibitor on various organ injuries is gaining attention. Regarding liver injury, Rho-kinase inhibitor is reported to prevent carbon tetrachloride (CCl4)- or dimethylnitrosamine-induced liver fibrosis and hepatic ischemia-reperfusion injury in rats. Because Rho-kinase inhibitor not only improved liver fibrosis but also reduced serum alanine aminotransferase (ALT) level in CCl4-induced liver fibrosis, we wondered whether Rho-kinase inhibitor might exert a direct hepatocyte-protective effect. We examined this possibility in acute CCl4 intoxication in rats. Rho-kinase inhibitor, HA-1077, reduced serum alanine ALT level in rats with acute liver injury induced by CCl4 with the improvement of histological damage and the reduction of the number of apoptotic cells. In cultured rat hepatocytes in serum-free condition, HA-1077 reduced apoptosis evaluated by quantitative determination of cytoplasmic histone-associated DNA oligonucleosome fragments with the reduction of caspase-3 activity and the enhancement of Bcl-2 expression. HA-1077 stimulated phosphorylation of Akt, and wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway, abrogated the reduction of hepatocyte apoptosis by HA-1077 in vitro. Furthermore, wortmannin abrogated the reduction of serum ALT level by HA-1077 in rats with acute liver injury induced by CCl4, suggesting that the activation of PI3-kinase/Akt pathway may be involved in the hepatocyte-protective effect by Rho-kinase inhibitor in vivo. In conclusion, Rho-kinase inhibitor prevented hepatocyte damage in acute liver injury induced by CCl4 in rats and merits consideration as a hepatocyte-protective agent in liver injury, considering its direct antiapoptotic effect on hepatocytes in vitro.     

Hepatotoxicity induced by diethylnitrosamine causes no significant disturbances of systemic glucose homeostasis in rats

In rats, a moderately hepatotoxic single dose of diethylnitrosamine (DEN) 100 mg/kg causing depletion of liver glycogen, elevation of aspartate aminotransferase and decreased liver uptake of 3-O-methylglucose, resulted in substantial changes in insulin and glucagon balance. Two days after DEN, insulin binding to liver membranes and insulin removal by the liver were sharply reduced whereas its binding to muscle and adipocyte membranes remained unaltered. Serum insulin (random and after an overnight fast) remained normal. Intravenous (I.V.) insulin (10 U/kg) caused the usual degree of hypoglycemia that, however, lasted longer than in the control animals. Removal of glucagon by liver was also depressed in spite of its normal binding to hepatocytes, and peripheral serum glucagon was increased three-fold. I.V. glucagon (40 micrograms/kg) resulted in a blunted response of plasma glucose. I.V. glucose tolerance test (1 g/kg) remained normal in spite of the insulin increase to a level twice as high as in the controls, and in spite of nonsuppressed glucagon. These changes were still present after 1-3 months, but disappeared by 6 months. The results demonstrate remarkable ability of homeostatic mechanisms to preserve normal plasma glucose and glucose tolerance in spite of dramatic changes in insulin and glucagon.     

Stimulation of lactase synthesis induced by starvation in the jejunum of adult rat

The effects of actinomycin D and of cycloheximide administration have been investigated on the enzyme activities of the jejunal brush border membrane in adult rats after a 48-hour period of starvation. The modifications in the protein and enzyme patterns of the brush border membrane and the incorporation of radiolabelled amino acid in the protein band corresponding to lactase have been studied in the nourished and in the starved animal. The results show that actinomycin D administration did not modify the stimulation of lactase activity caused by starvation whereas cycloheximide completely inhibited this process. The stimulation of lactase activity, in the starved animal, is related to a quantitative increase of the corresponding protein band and with enhanced incorporation of L-[3H]valine in this protein band after separation of brush border proteins by gel electrophoresis. It is concluded that the stimulation of lactase activity observed during starvation is the consequence of de novo synthesis of lactase molecules and that this process is regulated at a translational level. A general hypothesis is proposed in order to clear up partly the mechanism involved in the stimulation of lactase activity by food deprivation in the adult rat.     

Stimulation of rat hepatocyte proliferation in vitro and in vivo by factors derived from the bovine small intestinal mucosa

Summary A factor with a molecular weight of less than 1 kDa in the mucosa of the bovine small intestine (low molecular weight factor or LMW factor) stimulated DNA synthesis in rat hepatocytes in primary culture. This factor only showed its activity when it was added with a larger factor with a molecular weight of 30 kDa that was also found in the same tissue (high molecular weight factor or HMW factor). The LMW factor probably acts to enhance the action of a hepatotrophic growth factor, since EGF and HGF can substitute for the HMW factor. The action of the LMW factor was not due to the actions of low molecular weight substances such as norepinephrine, estradiol, triiodothyronine, and putrescine, which enhance the action of EGF or HGF, since substantial amounts of these substances were not found in the extract. When intraperitoneally administered into rats, after two-thirds hepatectomy, the LMW factor enhanced hepatocyte proliferation without the administration of the HMW factor. In the regenerating liver, a hepatotrophic growth factor(s), which acts synergistically with the LMW factor, might be properly provided, but the supply of the LMW factor might be below the level that maximally stimulates hepatocyte proliferation.     

Circadian synchronization of hepatocyte proliferation in young rats: the role played by adrenal hormones

Abstract. From the 20th day to the 30th day of life, the mitotic rhythm is progressively induced by a reduction in nocturnal values, while diurnal rhythms remain unchanged. Mitotic peaks emerge at 10.00 hours.
A labelling index wave occurs 8 hr before the corresponding mitotic wave, with a peak at 02.00 hours and a minimum in the evening, coincidental with the acrophase of plasma corticosterone level (activity phase).
Labelled mitoses curves and metaphase accumulation after colchicin injection show that the duration of the S, G₂ and M phases remain approximately constant and that the circadian variation is due to a variation in the rate of cells that enter these successive phases. During the synchronization period (from day 20 to 30), the growth fraction decreases progressively. Adrenalectomy at this time is followed by a higher cell proliferation and all rhythms disappear after 2 days.
Corticosterone injected before the triggering of the rhythmic activity in 17-day-old rats immediately reduces the labelling index, while the mitotic index is decreased 10 hr later; this delay is equal to the S + G₂ duration.
The results are discussed. They favour the hypothesis that the circadian variation of corticosterone is responsible for the induction of a circadian variation in developmental cell proliferation by inhibition of the G₁-S transition when it is higher in the evening.
The circadian rhythm of hepatic cell proliferation in rats appears on the 20th day of life, when the hypothalamo-adrenal axis is mature enough for circadian activity to occur.     

Circadian synchronization of hepatocyte proliferation in young rats: the role played by adrenal hormones

The circadian rhythm of hepatic cell proliferation in rats appears on the 20th day of life, when the hypothalamo-adrenal axis is mature enough for circadian activity to occur. From the 20th day to the 30th day of life, the mitotic rhythm is progressively induced by a reduction in nocturnal values, while diurnal rhythms remain unchanged. Mitotic peaks emerge at 10.00 hours. A labelling index wave occurs 8 hr before the corresponding mitotic wave, with a peak at 02.00 hours and a minimum in the evening, coincidental with the acrophase of plasma corticosterone level (activity phase). Labelled mitoses curves and metaphase accumulation after colchicine injection show that the duration of the S, G2 and M phases remain approximately constant and that the circadian variation is due to a variation in the rate of cells that enter these successive phases. During the synchronization period (from day 20 to 30), the growth fraction decreases progressively. Adrenalectomy at this time is followed by a higher cell proliferation and all rhythms disappear after 2 days. Corticosterone injected before the triggering of the rhythmic activity in 17-day-old rats immediately reduces the labelling index, while the mitotic index is decreased 10 hr later; this delay is equal to the S + G2 duration. The results are discussed. They favour the hypothesis that the circadian variation of corticosterone is responsible for the induction of a circadian variation in developmental cell proliferation by inhibition of the G1-S transition when it is higher in the evening.     

Biochemical microanalysis of alpha-glucosidase activity in preneoplastic and neoplastic hepatic lesions induced in rats by N-nitrosomorpholine

Preneoplastic and neoplastic hepatic lesions were induced in male Sprague-Dawley rats by oral administration of N-nitrosomorpholine (NNM) for 7 weeks at a concentration of 200 mg/l of drinking-water (stop model). Using a laser dissection technique and biochemical microanalysis, the activity of the lysosomal enzyme alpha-glucosidase was measured in glycogen storage foci emerging early, and in mixed or basophilic cell populations (foci and carcinomas) appearing later during hepatocarcinogenesis. In the liver tissue of normal appearance in both untreated controls and NNM-treated animals a slight gradient of alpha-glucosidase activity was observed leading from relatively high activities in zone 1 to lower activities in zone 3 of the liver lobule. In preneoplastic glycogen storage foci a considerable relative reduction in alpha-glucosidase activity was detected, suggesting that a decrease in the hydrolytic glycogen degradation contributes to the disturbance in phosphorylytic glycogen breakdown observed earlier in the majority of the glycogenotic foci. In contrast with glycogen storage foci, mixed and basophilic cell foci and particularly hepatocellular carcinomas showed a marked increase in alpha-glucosidase activity compared with that of normal liver tissue. The gradual enhancement in enzyme activity appeared to be closely related to the reduction in glycogen initially stored in excess during the later stages of hepatocarcinogenesis. The results support the concept that a fundamental shift in carbohydrate metabolism is characteristic of neoplastic transformation of hepatocytes.     

Impairment of long-term depression induced by chronic brain inflammation in rats

Although deficits in synaptic plasticity have been identified in aged or neuroinflamed animals with memory impairments, few studies have examined the cellular basis of plasticity in such animals. Here, we examined whether chronic neuroinflammation altered long-term depression (LTD) and studied the underlying mechanism of LTD impairment by neuroinflammation. Chronic neuroinflammation was induced by administration of lipopolysaccharide (LPS) to the fourth ventricle. Excitatory postsynaptic potentials were recorded extracellularly in the rat hippocampal CA1 area to examine alterations in synaptic plasticity. Chronic administration of LPS induced remarkable memory impairment in the Morris water maze test. N-methyl-d-aspartate receptor (NMDAR)-dependent LTD was almost absent in LPS-infused animals. The AMPA receptor (AMPAR)-mediated synaptic response was reduced in the LPS-infused group. These results suggest that reduction in NMDAR-dependent LTD might arise because of alterations in postsynaptic AMPARs as well as NMDARs and that such changes may be present in mild and early forms of Alzheimer-type dementia.     

Stimulation by vasopressin, angiotensin and oxytocin of gluconeogenesis in hepatocyte suspensions.

1. In hepatocytes from starved rats, vasopressin, angiotensin (angiotensin II) and oxytocin stimulated gluconeogenesis from lactate by 25--50%; minimal effective concentrations were about 0.02pM, 1 nM and 0.2 nM respectively. 2. Vasopressin and angiotensin also stimulated gluconeogenesis from alanine, pyruvate, serine and glycerol. EGTA decreased gluconeogenesis from these substrates. 3. Hormonal stimulation of gluconeogenesis from lactate was abolished in the absence of extracellular Ca2+. 4. Insulin did not prevent stimulation of gluconeogenesis by vasopressin or angiotensin. 5. The potency of the stimulatory effects of vasopressin and angiotensin on hepatic gluconeogenesis suggests they are operative in vivo. Also, the data suggest that Ca2+ plays a role in the stimulation by these hormones.     

Analysis of plasma tocopherols alpha,gamma, and 5-nitro-gamma in rats with inflammation by HPLC coulometric detection

Reactive nitrogen oxide species (RNOS) have been implicated as effector molecules in inflammatory diseases. There is emerging evidence that gamma-tocopherol (gammaT), the major form of vitamin E in the North American diet, may play an important role in these diseases. GammaT scavenges RNOS such as peroxynitrite by forming a stable adduct, 5-nitro-gammaT (NGT). Here we describe a convenient HPLC method for the simultaneous determination of NGT, alphaT, and gammaT in blood plasma and other tissues. Coulometric detection of NGT separated on a deactivated reversed-phase column was linear over a wide range of concentrations and highly sensitive (approximately 10 fmol detection limit). NGT extracted from blood plasma of 15-week-old Fischer 344 rats was in the low nM range, representing approximately 4% of gammaT. Twenty-four h after intraperitoneal injection of zymosan, plasma NGT levels were 2-fold higher compared to fasted control animals when adjusted to gammaT or corrected for total neutral lipids, while alpha- and gammaT levels remained unchanged. These results demonstrate that nitration of gammaT is increased under inflammatory conditions and highlight the importance of RNOS reactions in the lipid phase. The present HPLC method should be helpful in clarifying the precise physiological role of gammaT.     

Stimulation of intralysosomal proteolysis by cysteinyl-glycine, a product of the action of gamma-glutamyl transpeptidase on glutathione

The mechanism of the stimulatory effect of glutathione on proteolysis in mouse kidney lysosomes and a lack of an effect in lysosomes from the liver was investigated. The stimulation in kidney lysosomes was inhibited by serine plus borate, a reversibly inhibitor of gamma-glutamyl transpeptidase. Treatment of mouse kidney lysosome suspensions with L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin), an irreversibly inhibitor of the transpeptidase, also inhibited the effect of glutathione, but this inhibition was completely relieved by washing and addition of freshly prepared kidney membranes or purified gamma-glutamyl transpeptidase to the incubation mixtures. Cysteinyl-glycine, a product of the action of gamma-glutamyl transpeptidase, stimulated proteolysis in acivicin-inhibited kidney lysosome preparations similarly to glutathione, and cysteine had no effect at equivalent concentrations. Glutathione also stimulated proteolysis in liver lysosomes in the presence of washed kidney membranes or gamma-glutamyl transpeptidase, but the effect was similar to that produced by equivalent concentrations of cysteine. These results suggest that the stimulatory effect of glutathione was mediated by the action of gamma-glutamyl transpeptidase present in contaminating cell membrane fragments in the lysosome preparations, and that glutathione does not take part in intralysosomal proteolysis. However, the possibility that cysteinyl-glycine is a physiological intralysosomal disulfide reductant in kidney lysosomes has not been excluded.     

Regulation by calcium of the proliferation of heart cells from young adult rats

Summary Cells of primary and secondary cultures of rat heart ventricle were grown in a medium supplemented with homologous plasma to avoid the non-specific proliferogenic effects of foreign sera. Specific chelation of calcium from the medium arrested the cells' progression through their cycle at the G1 phase. A permanent or brief restoration of the extracellular calcium level (48 hr later) was followed, after a 5 to 6 hr delay, by a burst of DNA synthesis, and the resumption of mitotic activity. NRCC No. 14976.     

Stimulation of intralysosomal proteolysis by cysteinyl-glycine,a product of the action of γ-glutamyl transpeptidase on glutathione

The mechanism of the stimulatory effect of glutathione on proteolysis in mouse kidney lysosomes and a lack of an effect in lysomes from the liver was investigated. The stimulation in kidney lysosomes was inhibited by serine plus borate, a reversible inhibitor of γ-glutamyl transpeptidase. Treatment of mouse kidney lysosome suspensions with $\text{l}\text{-(αS,5S)-α-}\text{amino}\text{-3-}\text{chloro}\text{-4,5-}\text{dihydro}\text{-5-}\text{isoxazoleacetic}$ acid (acivicin), an irreversible inhibitor of the transpeptidase, also inhibited the effect of glutathione, but this inhibition was completely relieved by washing and addition of freshly prepated kidney membranes or purified γ-glutamyl transpeptidase to the incubation mixtures. Cysteinyl-glycine, a product of the action of γ-glutamyl transpeptidase, stimulated proteolysis in acivicin-inhibited kidney lysosome preparations similarly to glutathione, and cysteine had no effect at equivalent concentrations. Glutathione also stimulated proteolysis in liver lysosomes in the presence of washed kidney membranes or γ-glutamyl transpeptidase, but the effect was similar to that produced by equivalent concentrations of cysteine. These results suggest that the stimulatory effect of glutathione was mediated by the action of γ-glutamyl transpeptidase present in contaminating cell membrane fragments in the lysosome preparations, and that glutathione does not take part in intralysosomal proteolysis. However, the possibility that cysteinyl-glycine is a physiological intralysosomal disulfide reductant in kidney lysosomes has not been excluded.     

Morphometric and cytophotometric nuclear analysis of altered hepatocyte foci induced by N-nitrosomorpholine (NNM) and aflatoxin B1 (AFB1) in liver of Wistar rats

The progressive morphological changes in the liver during neoplastic transformation have been studied by histological, cytophotometric and morphometric methods in male Wistar rats treated with two carcinogens: N-nitrosomorpholine (NNM) and aflatoxin B1 (AFB1). Cytophotometric and morphometric analysis of hepatocyte nuclei using Feulgen-stained tissue sections were performed in morphologically normal hepatic parenchyma and in early preneoplastic foci composed of altered hepatocytes. Foci of clear cells, mixed cells and large basophilic cells possessed a ploidy distribution similar to the surrounding non-transformed parenchyma, while the small hyperbasophilic cell foci were predominantly diploid. These findings confirm that the foci composed of PAS-negative, small hyperbasophilic cells with an unique diploid content may represent one of the earliest stages in the neoplastic transformation.     

Silibinin ameliorates arsenic induced nephrotoxicity by abrogation of oxidative stress, inflammation and apoptosis in rats

Arsenic (As) is an environmental and industrial pollutant that affects various organs in human and experimental animals. Silibinin is a naturally occurring plant bioflavonoid found in the milk thistle of Silybum marianum, which has been reported to have a wide range of pharmacological properties. A body of evidence has accumulated implicating the free radical generation with subsequent oxidative stress in the biochemical and molecular mechanisms of As toxicity. Since kidney is the critical target organ of chronic As toxicity, we carried out this study to investigate the effects of silibinin on As-induced toxicity in the kidney of rats. In experimental rats, oral administration of sodium arsenite [NaAsO₂, 5?mg/(kg?day)] for 4?weeks significantly induced renal damage which was evident from the increased levels of serum urea, uric acid, creatinine with a significant (p?<?0.05) decrease in creatinine clearance. As also significantly decreased the levels of urea, uric acid and creatinine in urine. A markedly increased levels of lipid peroxidation markers (thiobarbituric acid reactive substances and lipid hydroperoxides) and protein carbonyl contents with significant (p?<?0.05) decrease in non-enzymatic antioxidants (total sulfhydryl groups, reduced glutathione, vitamin C and vitamin E) and enzymatic antioxidants (superoxide dismutase, catalase, glutathione peroxidase and glutathione S-transferase), Glutathione metabolizing enzymes (glutathione reductase and glutathione-6-phosphate dehydrogenase) and membrane bound ATPases were also observed in As treated rats. Co-administration of silibinin (75?mg/kg?day) along with As resulted in a reversal of As-induced biochemical changes in kidney accompanied by a significant decrease in lipid peroxidation and an increase in the level of renal antioxidant defense system. The histopathological and immunohistochemical studies in the kidney of rats also shows that silibinin (75?mg/kg?day) markedly reduced the toxicity of As and preserved the normal histological architecture of the renal tissue, inhibited the caspase-3 mediated tubular cell apoptosis and decreased the NADPH oxidase, iNOS and NF-κB over expression by As and upregulated the Nrf2 expression in the renal tissue. The present study suggests that the nephroprotective potential of silibinin in As toxicity might be due to its antioxidant and metal chelating properties, which could be useful for achieving optimum effects in As-induced renal damage.