Optimal conditions for cordycepin production in surface liquid-cultured Cordyceps militaris treated with porcine liver extracts for suppression of oral cancer.

Cordycepin is one of the most crucial bioactive compounds produced by Cordyceps militaris and has exhibited antitumor activity in various cancers. However, industrial production of large amounts of cordycepin is difficult. The porcine liver is abundant in proteins, vitamins, and adenosine, and these ingredients may increase cordycepin production and bioconversion during C. militaris fermentation. We observed that porcine liver extracts increased cordycepin production. In addition, air supply (2 h/d) significantly increased the cordycepin level in surface liquid-cultured C. militaris after 14 days. Moreover, blue light light-emitting diode irradiation (16 h/d) increased cordycepin production. These findings indicated that these conditions are suitable for increasing cordycepin production. We used these conditions to obtain water extract from the mycelia of surface liquid-cultured C. militaris (WECM) and evaluated the anti-oral cancer activity of this extract in vitro and in vivo. The results revealed that WECM inhibited the cell viability of SCC-4 oral cancer cells and arrested the cell cycle in the G2/M phase. Oxidative stress and mitochondrial dysfunction (mitochondrial fission) were observed in SCC-4 cells treated with WECM for 12 hours. Furthermore, WECM reduced tumor formation in 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis through the downregulation of proliferating cell nuclear antigen, vascular endothelial growth factor, and c-fos expression. The results indicated that porcine liver extracts irradiated with blue light light-emitting diode and supplied with air can be used as a suitable medium for the growth of mycelia and production of cordycepin, which can be used in the treatment of oral cancer.

[DIFFERENTIATION OF HUMAN UMBILICAL CORD MESENCHYMAL STEM CELLS INTO HEPATOCYTES INDUCED BY RAT FIBROTIC LIVER TISSUE EXTRACTS].

OBJECTIVE: To investigate the differentiation potential of human umbilical cord mesenchymal stem cells (HUCMSCs) into hepatocytes induced by rat fibrotic liver tissue extracts. METHODS: Liver fibrosis was induced in the Sprague Dawley rats (weighting, 180-220 g) by repeated intraperitoneal injections of 3% thioacetamide-saline at a dose of 200 mg/kg twice a week for 4 weeks; fibrotic liver tissues were used to prepare liver homogenate supernatants. The HUCMSCs at passage 3 were cultured in DMEM/F12 with 10% fetal bovine serum (FBS) (control group) and in DMEM/F12 with 10% FBS and 50 g/L liver homogenate supernatants (experimental group) for 7 days. The morphological changes of the cells were recorded; the protein levels of cytokeratin 18 (CK18), alpha fetoprotein (AFP), and CYP3A4 were measured using Western blot. The glycogen storing ability of the cells was detected by periodic acid-schiff (PAS) staining. Furthermore, the synthesis of albumin (ALB) and blood urea nitrogen (BUN) was measured. RESULTS: In experimental group, after 1 day of induction, the stem cells of fusiform shape began to lose sharp edges and progressively shrunk, and then they changed into hepatocyte-like cells with round and irregular shape at 7 days. Positive expressions of AFP, CK18, and CYP3A4 were observed in the experimental group, but negative expression in the control group. The concentrations of BUN and ALB were (0.43 ± 0.07) mmol/L and (8.08 ± 0.41) µg/mL in the control group and were (2.52 ± 0.20) mmol/L and (41.48 ± 4.11) µg/mL in the experimental group, showing significant differences (t=24.160, P = 0.000; t = 19.810, P = 0.000). PAS staining results showed navy blue nucleus and lavender cytoplasm in the control group, but dark purple cell body and visible nucleus in the experimental group. CONCLUSION: HUCMSCs could differentiate into hepatocyte-like cells induced by rat fibrotic liver tissue extracts, which have hepatocyte biomarkers (AFP, CK18, and CYP3A4) and hepatocyte-specific functions of glycogen storage, urea production and ALB secretion, so they could partially replace the function of hepatocytes, that may be one of the therapeutic mechanisms of stem cell transplantation.

A comparison of the chemical and liver extract-induced hepatic differentiation of adipose derived stem cells.

Adipose-derived stem cells (ADSCs) have been put forward as promising therapeutics for end-stage liver disease (ESLD). In the present study, we compared the effects of defined chemicals and liver extract on the hepatic differentiation of ADSCs. ADSCs were isolated according to the method described in our previously published study. Subsequently, the differentiation of ADSCs was induced separately by chemicals (including hepatic growth factor (HGF), fibroblast growth factor (FGF), and oncostatin M (OSM)) and liver extract (30 µg/ml) in a total period of 21 d. The efficiency of hepatic differentiation was evaluated by changes in the cell morphology, gene expression, and cellular function. The results showed that the liver extract promoted the hepatic differentiation of ADSCs to a significantly greater extent than the chemicals. In the group of ADSCs treated with liver extract, changes in the cell morphology began sooner, and the expression of alpha-FP and albumin genes was higher than that in the chemically treated group. The ADSCs in both the groups stained positive for anti-alpha trypsin (AAT) and albumin markers. The cells also exhibited glycogen storage capacity. Therefore, we concluded that the liver extract could efficiently induce the differentiation of ADSCs into hepatocyte-like cells. This study reveals the potential of mesenchymal stem cell differentiation in the liver extract, which supports further preclinical and clinical research on the application of ADSCs in ESLD treatment.

Genotoxicity evaluation of sesamin and episesamin.

Sesamin is a major lignan that is present in sesame seeds and oil. Sesamin is partially converted to its stereoisomer, episesamin, during the refining process of non-roasted sesame seed oil. We evaluated the genotoxicity of these substances through the following tests: a bacterial reverse mutation assay (Ames test), a chromosomal aberration test in cultured Chinese hamster lung cells (CHL/IU), a bone marrow micronucleus (MN) test in Crlj:CD1 (ICR) mice, and a comet assay using the liver of Sprague-Dawley (SD) rats. Episesamin showed negative results in the Ames test with and without S9 mix, in the in vitro chromosomal aberration test with and without S9 mix, and in the in vivo comet assay. Sesamin showed negative results in the Ames test with and without S9 mix. In the in vitro chromosomal aberration test, sesamin did not induce chromosomal aberrations in the absence of S9 mix, but induced structural abnormalities at cytotoxic concentrations in the presence of S9 mix. Oral administration of sesamin at doses up to 2.0g/kg did not cause a significant increase in either the percentage of micronucleated polychromatic erythrocytes in the in vivo bone marrow MN test or in the % DNA in the comet tails in the in vivo comet assay of liver cells. These findings indicate that sesamin does not damage DNA in vivo and that sesamin and episesamin have no genotoxic activity.

Production of erythriod cells from human embryonic stem cells by fetal liver cell extract treatment.

BACKGROUND: We recently developed a new method to induce human stem cells (hESCs) differentiation into hematopoietic progenitors by cell extract treatment. Here, we report an efficient strategy to generate erythroid progenitors from hESCs using cell extract from human fetal liver tissue (hFLT) with cytokines. Human embryoid bodies (hEBs) obtained of human H1 hESCs were treated with cell extract from hFLT and co-cultured with human fetal liver stromal cells (hFLSCs) feeder to induce hematopoietic cells. After the 11 days of treatment, hEBs were isolated and transplanted into liquid medium with hematopoietic cytokines for erythroid differentiation. Characteristics of the erythroid cells were analyzed by flow cytometry, Wright-Giemsa staining, real-time RT-PCR and related functional assays. RESULTS: The erythroid cells produced from hEBs could differentiate into enucleated cells and expressed globins in a time-dependent manner. They expressed not only embryonic globins but also the adult-globin with the maturation of the erythroid cells. In addition, our data showed that the hEBs-derived erythroid cells were able to act as oxygen carriers, indicating that hESCs could generate functional mature erythroid cells. CONCLUSION: Cell extract exposure with the addition of cytokines resulted in robust erythroid -like differentiation of hEBs and these hEBs-derived erythroid cells possessed functions similar to mature red blood cells.

Study on the antidiabetic mechanism of a shark liver peptide, S-8300, in alloxan-induced diabetes.

OBJECTIVES: The aim was to evaluate the antidiabetic mechanism of S-8300 in alloxan-diabetic mice. METHODS: Diabetes was induced by a single intravenous injection of alloxan (60 mg/kg). The effects of S-8300 on diabetic mice were investigated by observing the change in fasting plasma glucose, detecting Fas mRNA by reverse transcriptase-polymerase chain reaction, Fas protein expression in the pancreas by immunohistochemistry and Western blot, and the DNA fragmentation pattern forming a ladder by electrophoresis. KEY FINDINGS: A significant decrease in fasting plasma glucose was observed, and Fas mRNA and Fas protein expression in the pancreas were attenuated in diabetic mice treated with S-8300. Treatment with S-8300 also attenuated DNA ladder formation. CONCLUSIONS: The results suggest that S-8300 inhibits Fas protein-mediated apoptosis of pancreas cells.

Cells extract from fetal liver promotes the hematopoietic differentiation of human embryonic stem cells.

Here, we have now developed a new inducing system to promote the differentiation of human stem cells (hESCs) toward hematopoietic lineages by the treatment with cells extract of human fetal liver tissue (hFLT). The embryoid bodies (EBs) obtained from human H1 embryonic stem cells were exposed to buffer, hFLT cells extract, heated hFLT cell extract, and cell extract of human liver cells lines-LO2. Then, the feature of EBs in different groups was characterized by real-time RT-PCR and colony-forming assays. The results showed the treatment by hFLT cells extract could activate the hematopoietic genes expression and improve the capacity for hematopoietic progenitor development of hEBs. After that, we cocultured hFLT extract treated hEBs on the hFLSCs (human fetal liver stromal cells) feeder to differentiate them into hematopoietic cells. As a control, untreated hEBs were cocultured on hFLSCs feeder with cytokines. The feature of induced cells from hEBs was characterized by flow cytometry, Wright-Giemsa staining, and colony-forming assays. The results demonstrated that hFLT cells extract was capable of inducing hEBs into hematopoietic cells and combing it with hFLSCs feeder could largely promote hematopoietic differentiation of hESCs. This method may supply a new way to substitute the cytokines required in hematopoietic induction of hESCs.

Dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA), two naturally occurring benzoxazinones contained in sprouts of Gramineae are potent aneugens in human-derived liver cells (HepG2).

Benzoxazinoids (BAs) are toxic constituents of sprouts of Gramineae such as wheat, maize and rye and are part of the plant defence system against pests. In the last years, sprouts have been increasingly consumed as health foods and are also used for the production of dietary supplements. In the present study we investigated the mutagenic activities of the two most abundant BAs, namely 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) in the Salmonella/microsome assay and additionally, in micronucleus (MN) assay and single cell gel electrophoresis (SCGE) assay in a human-derived liver cell line (HepG2). DIBOA caused significant induction of his(+) revertants in all three strains in the range between 0.02 and 0.50 mg/plate; the highest activity was observed in TA100 (fivefold increase over the background at the highest dose level). The effect in YG1024 (a derivative of TA98 with increased acetyltransferase activity) was only slightly higher than the effect in the parental strain indicating that acetylation plays no crucial role in the activation of this BA. DIMBOA was in general less active and a positive result was only seen in the base substitution strain (TA100). Addition of rat liver homogenate (S9-mix) led to a significant (ca. twofold) increase of the mutagenic activities of both BAs. In SCGE assays with HepG2 cells consistently negative results were obtained with both compounds whereas in MN assays significant dose dependent effects were observed under similar experimental conditions. DIMBOA caused significant effects already at concentrations > or =1 microM; at the highest dose (20 microM) the MN frequency was sevenfold higher than the background level. DIBOA caused weaker effects and was positive at doses > or =2.5 microM, the maximal induction (twofold over background) was observed with 20 microM. Overall, DIMBOA was ca. 30-fold more active as DIBOA. Subsequent experiments with pancentromeric probes showed that >80% of the MN induced at the highest doses gave a centromere positive signal indicating that both BAs are aneugenic. This is an interesting observation as it is assumed that aneuploidy is a key event in cancer induction and at present no other aneugenic plant-derived substances of dietary relevance are known.

Differentiation of human umbilical cord blood stem cells into hepatocytes in vivo and in vitro.

AIM: To study the condition and potentiality of human umbilical cord blood stem cells (HUCBSC) to differentiate into hepatocytes in vivo or in vitro. METHODS: In a cell culture study of human umbilical cord blood stem cell (HUCBSC) differentiation, human umbilical cord blood mononuclear cells (HUCBMNC) were separated by density gradient centrifugation. Fibroblast growth factor (FGF) and hepatocyte growth factor (HGF) and the supernatant of fetal liver were added in the inducing groups. Only FGF was added in the control group. The expansion and differentiation of HUCBMNC in each group were observed. Human alpha fetoprotein (AFP) and albumin (ALB) were detected by immunohistochemistry. In the animal experiments, the survival SD rats with acute hepatic injury after carbon tetrachloride (CCL4) injection 48 h were randomly divided into three groups. The rats in group A were treated with human umbilical cord blood serum. The rats in group B were treated with HUCBMNC transplantation. The rats in group C were treated with HUCBMNC transplantation followed by intraperitoneal cyclophosphamide for 7 d. The rats were killed at different time points after the treatment and the liver tissue was histopathologically studied and human AFP and ALB detected by immunohistochemistry. The human X inactive-specific transcript gene fragment in the liver tissue was amplified by PCR to find human DNA. RESULTS: The results of cell culture showed that adherent cells were stained negative for AFP or ALB in control group. However, the adherent cells in the inducing groups stained positive for AFP or ALB. The result of animal experiment showed that no human AFP or ALB positive cells present in the liver tissue of group A (control group). However, many human AFP or ALB positive cells were scattered around sinus hepaticus and the central veins of hepatic lobules and in the portal area in group B and group C after one month. The fragment of human X chromagene could be detected in the liver tissue of groups B and C, but not in group A. CONCLUSION: Under certain conditions HUCBSC can differentiate into liver cells in vivo and in vitro.

Antidiabetic effect of a new peptide from Squalus mitsukurii liver (S-8300) in alloxan-diabetes.

1. The purpose of the present study was to evaluate the antidiabetic effect of S-8300 in alloxan diabetic mice. 2. Diabetes was induced by single intravenous injection of alloxan (60 mg/kg bodyweight). 3. The effects of S-8300 on diabetic mice were investigated by observing changes in the glycometabolic index (fasting plasma glucose, glycosylated haemoglobin), the lipometabolic index (triglyceride, cholesterol, free fatty acids), anti-oxidant index (superoxide dismutase, malondialdehyde) and in the degree of injury of beta-cells in the pancreatic islets. 4. A significant decrease in fasting plasma glucose, glycosylated haemoglobin, triglycerides, cholesterol, free fatty acids in the plasma and malondialdehyde in the tissues, as well as a significant increase in the activity of superoxide dismutase, was observed in diabetic mice treated with 10, 3 and 1 mg/kg S-8300. 5. Treatment with S-8300 also attenuated the degree of injury of beta-cells. 6. A comparison was made between the action of S-8300 and 6 U/kg insulin. The effects of S-8300 were similar to those of insulin.

The induction of gene mutations and micronuclei by oxiranes and siloranes in mammalian cells in vitro.

Oxiranes and siloranes are candidate molecules for the development of composite materials with low shrinkage. Since some of these molecules are highly reactive, they could lead to adverse biological effects from underlying genetic mechanisms. Therefore, we analyzed the formation of micronuclei (chromosomal aberrations) and the induction of gene mutations (HPRT assay) in mammalian cells. The numbers of micronuclei induced by the oxirane di(cyclohexene-epoxidemethyl)ether (Eth-Ep) at low concentrations (10 micro M) were about five-fold higher than controls. The related compound epoxy cyclohexyl methyl-epoxy cyclo-hexane carboxylate (Est-Ep) was less effective. The activity of diglycidylether of bisphenol A (BADGE) was even lower but similar to the most reactive silorane, di-3,4-epoxy cyclohexylmethyl-dimethyl-silane (DiMe-Sil). No induction of micronuclei was detected in the presence of a rat liver homogenate (S9). Est-Ep and Eth-Ep also induced gene mutations. Our analyses indicated low mutagenic potentials of siloranes; however, some oxiranes induced strong effects at two genetic endpoints.

Mutant spectra analysis at hisG46 in Salmonella typhimurium strain YG1029 induced by mammalian S9- and plant-activated aromatic amines.

Mutant spectra analysis was conducted with spontaneous hisG46 revertants of Salmonella typhimurium strain YG1029 and revertants induced by the plant- and mammalian S9-activation of benzidine and 4-aminobiphenyl (4-ABP). Under preincubation conditions, YG1029 cells were exposed to benizidine or 4-ABP with mammalian S9 activation or to a high molecular weight fraction that contained the plant-activated products. The induced revertants were isolated at mutagen concentrations that caused an increased mutant frequency of approximately 4- to 10-fold above background. Genomic DNA from each revertant was isolated and the hisG region was amplified using polymerase chain reaction (PCR). Using a series of specific probes and a modified version of the ECL3's-oligolabelling and detection system, each of the six possible base-pair substitution mutations at hisG46 that leads to a reversion event was determined. Of the YG1029 spontaneous revertants, transition mutations were 31.8% and transversion mutations were 68.2%. The YG1029 spontaneous mutant spectrum differed significantly from the spontaneous spectrum of TA1535 but did not significantly differ from the spontaneous TA100 mutant spectrum. The differences of the spontaneous mutant spectra among these highly related strains illustrate that the introduction of the plasmid pKM101 into S. typhimurium increased the frequency of transversions (CCC-->ACC; CCC-->CAC) and reduced site 2 (CCC-->CTC) transitions. With plant-activated benzidine, 21.1% of recovered revertants resulted from transitions and 78.9% from transversions while S9 activated-benzidine induced revertants were recovered as 14.2% from transition and 85.8% from transversion mutations. Plant-activated 4-ABP recovered 20.0% transitions and 80.0% transversions. S9-activated 4-ABP-induced 21.4% transitions and 78.6% transversions. Chi-square analysis of mutant spectra indicated that the DNA lesions that resulted in reversion at the hisG46 allele induced by plant-activated benzidine or 4-ABP were different from those generated after mammalian S9 activation of these promutagens. The plant-activated benzidine and 4-ABP induced statistically identical mutant spectra. Also, the mammalian-activated benzidine and 4-ABP induced statistically similar mutant spectra. These data show that the plant-activated and mammalian-activated aromatic amine products inflicted different types or distributions of DNA lesions that were reflected in the resulting induced mutant spectra.

Antiperoxidative, anti-inflammatory, and antimutagenic activities of ethanol extract of the mycelium of Ganoderma lucidum occurring in South India.

Free radical mediated genetic instability is widely thought to be a major etiological factor for initiation of carcinogenesis. Mushrooms represent a largely untapped source of powerful new pharmaceutical products. In the present study, we examined the antiperoxidative, anti-inflammatory, and antimutagenic activities of the ethanol extract of the mycelium of a medicinal mushroom, Ganoderma lucidum, occurring in south India. Antiperoxidative activity was evaluated using Fe(2+)-ascorbate-induced lipid peroxidation in rat liver homogenate and a phorbol ester (croton oil)-induced lipid peroxidation in mouse skin. Antiinflammatory activity was evaluated against carrageenan-induced acute and formalin-induced chronic inflammatory paw edema in mouse and phorbol ester-induced mouse skin inflammation. Antimutagenic activity was determined by the Ames mutagenicity assay using histidine mutant of Salmonella typhimurium strains TA 98, TA100, and TA102. Sodium azide (NaN(3)), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NPD), and benzo[a]pyrene (B[a]P) were used as the mutagens. The extract showed significant inhibition of Fe(2+)-induced peroxidation of lipid in rat liver (IC(50) 510 +/- 22 microg/ml) and 37% inhibition of croton oil-induced peroxidation on the mouse skin at 20 mg/0.1 ml/skin. Carrageenan-induced acute and formalin-induced chronic inflammatory edema were inhibited by 56 and 60%, respectively, by the extract at 1,000 mg/kg body wt (i.p). The extract at a concentration of 5 mg/plate showed inhibition of mutagenicity elicited by direct acting mutagens, NaN(3) (55.5 and 75.7%) and MNNG (50.0 and 57.5%) for S. typhymurium strains TA100 and TA102, respectively. The extract at the same concentration also inhibited mutagenicity elicited by NPD (52.4 and 64.2%) and B[a]P (60.7 and 59.6%) for TA98 and TA100 strains, respectively. The B[a]P was activated in the presence of rat liver microsomal (S9) fraction. The results of our study revealed that ethanol extract of Ganoderma lucidum mycelium possessed significant antiperoxidative, antiinflammatory, and antimutagenic activities. The findings suggest a medicinal use for the ethanol extract of the mycelium of G. lucidum occurring in South India.

Detection, characterisation and purification of a murine liver factor capable of desensitising towards the lethal activity of tumour necrosis factor.

Tumour necrosis factor (TNF) is a major mediator in septic shock and several inflammatory diseases such as hepatitis. Galactosamine (GalN) sensitises experimental animals for TNF and the combination TNF/GalN leads to a lethal inflammatory hepatitis. We describe that a single injection of lipopolysaccharide (LPS), interleukin-1 (IL-1) or TNF can desensitise against the lethality induced by TNF/GalN, but also against changes in metabolic parameters such as hypothermia and transaminase release, in a dose responsive way. We also describe the desensitising capacity of a component present in Mouse Liver Extract (MLE). The MLE desensitises mice against the effects of TNF/GalN in a dose responsive way. The activity of the MLE is heat labile and does not involve LPS, TNF, IL-1 or TNF soluble receptors. We describe partial and complete purification of the factor. Partially pure material protects mice against all changes induced by TNF/GalN. The protection is dose dependent and heat labile and also possible in endotoxin-hyporesponsive C3H/HeJ mice. The pure material protects against lethality, hypothermia and AST release and it appears as a heat labile protein of relative molecular weight of 70 kDa probably with a break down product of 35 kDa.

Toxicity, mutation frequency and mutation spectrum induced by dacarbazine in CHO cells expressing different levels of O(6)-methylguanine-DNA methyltransferase.

The toxicity and mutagenicity (including the mutation spectrum induced) of dacarbazine, a methylating cytostatic drug, was examined in CHO cells expressing different levels of the repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT). Expression of low or high levels of a transfected human MGMT gene under the control of the metallothionein promoter protected the cells against dacarbazine-induced toxicity and mutagenesis. In the absence of MGMT expression, the mutation spectrum in the HPRT locus was dominated by GC-->AT transitions (mostly found at 5'Pu-G sequences), while there were also a few AT-->GC transitions. Expression MGMT was associated with a substantial decrease of GC-->AT mutations, suggesting that these mutations arose primarily via O(6)-methylguanine. These data illustrate the important role of the latter lesion in the drug's mutagenic and cytotoxic activity.

Effect of cod-liver oil extract on the buccal permeation of ergotamine tartrate.

Ergotamine tartrate (ET) is used clinically in the treatment of migraines. However, the bioavailability of ET is rather poor following oral administration. Therefore, we tried to improve ET delivery using buccal administration. The purpose of this study was to investigate the characteristics of the permeation of ET through the hamster cheek pouch in vitro using a two-chamber diffusion cell, and to evaluate the effect of permeation enhancers on the transbuccal delivery of ET. Cod-liver oil extract (CLOE), polyoxyethylene hydrogenated castor oil (HCO 60), sodium glycocholate (GC), and sodium caprate (CA) were selected as premeation enhancers considering their low irritancy of the mucosa. When the enhancers were added to the donor cell at a 5% concentration each, the ET permeation rate markedly increased compared with that in a control not containing enhancer. Among these enhancers, CLOE exhibited the greatest effect. Because CLOE is composed of 16 kinds of fatty acids, the enhancement action of each of the major components was separately determined. As major fatty acids, palmitic acid, oleic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) were selected and their enhancing effects were studied. The enhancing effect of each fatty acid was significantly lower than that of CLOE.

Induction of hepatocyte proliferation and prevention of hepatocyte apoptosis by phenobarbital related to local humoral factor in mouse liver.

AIM: To study the association of phenobarbital (Phe) inducing hepatocyte proliferation and blocking hepatocyte apoptosis with local humoral factor in liver. METHODS: The ratio of liver/body weight, DNA content, regressive rate of hyperplastic liver, and DNA fragmentation were used to investigate whether the Phe-treated mouse liver extract (PMLE) and PMLE-95 (PMLE heated at 95 degrees C for 30 min) possessed Phe-like effects on mouse liver. Meantime, the effects of pretreatment with trypsin, RNAase, and DNAase on the activity of PMLE-95 were observed, and the differences of components between PMLE-95 and NMLE-95 (normal mouse liver extract, NMLE heated at 95 degrees C for 30 min) were analyzed with HPLC. RESULTS: PMLE-95 stimulated hepatocyte proliferation and prevented hepatocyte apoptosis caused by withdrawing Phe in mice, and the activity of PMLE-95 was eliminated after the pretreatment with trypsin. On the chromatograms PMLE-95 had 5 main peaks, while NMLE-95 had only 4 peaks. CONCLUSION: The effects of Phe on the liver were mediated by an intrinsic protein or peptide substance produced in response for the stimulation of Phe in mouse liver.

Effect of tissue and plasma factors on kidney proliferation.

Liver extract, plasma from intact mice, ES2 tumour extract and plasma from tumour bearing mice has an inhibiting effect on the mitotic activity of hepatocytes and duodenal enterocytes. In the present experiments, the effect of these treatments on the mitotic activity of renal tubular cells was studied. C3HS 28 day-old male mice, standardized for periodicity analysis were used. The determination of normal mitotic circadian curve of the renocytes was done. A second batch of mice were injected with 0.01 ml/gr of either liver extract, plasma from intact mice, ES2 tumour extract or plasma from tumour bearing mice, at 16:00 hours and controlled at 08:00, 12:00 and 16:00 hs during 2 consecutive days post treatment. Colchicine (2 micrograms/gr) was injected 4 hours before killing. Kidneys were processed for histology and mitotic index determinations. Results were expressed as colchicine metaphases per 1000 nuclei, and showed that mitotic activity values of treated animals were significantly lower than those of controls. In conclusion, mitotic activity inhibition of renocytes may be due to some non specific plasmatic and/or tissue factors.

Effect of tissue and plasma factors on kidney proliferation

Liver extract, plasma from intact mice, ES2 tumour extract and plasma from tumour bearing mice has an inhibiting effect on the mitotic activity of hepatocytes and duodenal enterocytes. In the present experiments, the effect of these treatments on the mitotic activity of renal tubular cells was studied. C3HS 28 day-old male mice, standardized for periodicity analysis were used. The determination of normal mitotic circadian curve of the renocytes was done. A second batch of mice were injected with 0.01 ml/gr of either liver extract, plasma from intact mice, ES2 tumour extract or plasma from tumour bearing mice, at 16:00 hours and controlled at 08:00, 12:00 and 16:00 hs during 2 consecutive days post treatment. Colchicine (2 micrograms/gr) was injected 4 hours before killing. Kidneys were processed for histology and mitotic index determinations. Results were expressed as colchicine metaphases per 1000 nuclei, and showed that mitotic activity values of treated animals were significantly lower than those of controls. In conclusion, mitotic activity inhibition of renocytes may be due to some non specific plasmatic and/or tissue factors.