Rabbit Liver Homogenates Research Articles

A New Avenue for Enhanced Treatment of Hyperuricemia and Oxidative Stress: Design, Synthesis and Biological Evaluation of Some Novel Mutual Prodrugs Involving Febuxostat Conjugated with Different Antioxidants

Febuxostat (FEB) is the first non-purine xanthine oxidase inhibitor (XOI) used for the treatment of hyperuricemia and gout. The oxidative stress induced by reactive oxygen species (ROS) which accompany purine metabolism by XO, could contribute to cellular damage and several pathological conditions. In this view, the present work addresses the evaluation of combining the hypouricemic effect of FEB and the free radical scavenging potential of various natural antioxidants in a single chemical entity by implementing the “mutual prodrug” strategy. Accordingly, a series of five ester prodrugs containing FEB together with different naturally occurring antioxidants namely, thioctic acid (4), thymol (5), menthol (6), vanillin (7), and guaiacol (8) was synthesized. Prominently, all the chemically conjugated prodrugs (4 – 8) revealed an obvious increase in the hypouricemic and antioxidant potentials when compared with their corresponding promoieties and physical mixtures. Moreover, they showed a potential protective effect against CCl4-induced hepatotoxicity and oxidative stress, together with no cytotoxicity on normal breast cells (MCF10A). Furthermore, the in vitro chemical and enzymatic stability studies of the prodrugs (4 – 8) using a developed HPLC method, verified their stability in different pHs, and rapid hydrolysis in rabbit plasma and liver homogenate to their parent metabolites. Moreover, the prodrugs (4 – 8) showed higher lipophilicity and lower aqueous solubility when compared to the parent drugs. Finally, the obtained merits from the implementation of the mutual prodrug strategy would encourage further application in the development of promising candidates with high therapeutic efficacy and improved safety profiles.

ELISA for detection of variant rabbit haemorrhagic disease virus RHDV2 antigen in liver extracts

The emergence and rapid spread of variant of the rabbit hemorrhagic disease virus (RHDV2) require new diagnostic tools to ensure that efficient control measures are adopted.In the present study, a specific sandwich enzyme-linked immunosorbent assay (ELISA) for detection of RHDV2 antigens in rabbit liver homogenates, based on the use of an RHDV2-specific monoclonal antibody (Mab) 2D9 for antigen capture and an anti-RHDV2 goat polyclonal antibody (Pab), was developed. This ELISA was able to successfully detect RHDV2 and RHDV2 recombinant virions with high sensitivity (100%) and specificity (97.22%). No cross-reactions were detected with RHDV G1 viruses while low cross-reactivity was detected with one of the RHDVa samples analyzed. The ELISA afforded good repeatability and had high analytical sensitivity as it was able to detect a dilution 1:163,640 (6.10ng/mL) of purified RHDV-N11 VLPs, which contained approximately 3.4×108molecules/mL particles.The reliable discrimination between closely related viruses is crucial to understand the epidemiology and the interaction of co-existing pathogens. In the work described here we design and validate an ELISA for laboratory based, specific, sensitive and reliable detection of RHDVb/RHDV2. This ELISA is a valuable, specific virological tool for monitoring virus circulation, which will permit a better control of this disease.

Fast specific field detection of RHDVb

This work describes a simple and rapid test for field detection of the emerging rabbit pathogen RHDVb. The assay is specific for RHDVb, showing no cross-reactivity with other RHDV types giving a specific result in under 10min using rabbit liquid exudates or liver homogenate samples taken at necropsy.

Acute and sub-lethal toxicological evaluation of flavonoid fraction of <i>Monodora myristica</i> Geartn Dunnel seeds

This study investigated acute toxicity of flavonoid fraction of Monodora myristica in mice and sub lethal toxicity in rabbits. Powdered seed of M. myristica (312 g) was extracted with 2.5 L of 70% (v/v) ethanol for 24 h followed by concentration under reduced pressure at 40 oC to obtain a brown residue termed ethanolic extract (EE). The EE was subjected to solvent-solvent partitioning with ethyl acetate to obtain the flavonoid fraction (FF). The acute toxicity test involved oral administration of graded doses of FF of M. myristica to six groups of mice. Biochemical changes as a result of sub lethal exposure of FF to rabbits were investigated at 500 mg/kg bwt and 1000 mg/kg bwt for 28 days. The effects of the extract of M. myristica on total protein, albumin, bilirubin, creatinine, urea, as well as the activities on specific marker enzymes (L-alanine and Laspartate aminotransferases, alkaline and acid phosphatases and gamma glutamyltransferase) were evaluated in the plasma and liver homogenates of rabbits. The histopathological evaluation of the liver, brain, heart, lungsand kidney of rabbits were carried out. FF of M. myristica was observed to be non toxic in mice with LD50 greater than 5000 mg/kg bwt. There were no significant differences (p > 0.05) in the total protein, albumin, bilirubin, creatinine, uric acid, urea levels in the liver and plasma of treated animals. Moreover, some activities of specific marker enzymes such as L-alanine and L-aspartate aminotransferases, acid and alkaline phosphatases and gamma glutamytransferase were not adversely affected. Histopathological findings showed no significant differences between control and the treated groups and revealed that the brain, lungs, heart, liver and kidney tissues of rabbit were not adversely affected.Keywords: Ethanolic extracts (EE), flavonoid fraction (FF), toxicity, Monodora myristica (MM).

Free Radical Scavenging and In-vitro Antioxidant Effects of Ethanol Extract of the Medicinal Herb Chromolaena odorata Linn.

The ethanol extract of the leaf of Chromolaena odorata (Linn) was assessed for free-radical-scavenging and antioxidant potentials. Ability of the extract to scavenge reactive intermediates (superoxide ion O2·-, hydrogen peroxide H2O2, nitric oxide NO˙, hydroxyl radical OH˙) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, were used to assess its free radical scavenging potentials. Antioxidant potential was studied by assessing in-vitro inhibition of lipid peroxidation in both the brain (Neuro-protective potentials) and liver homogenates of Fenton-oxidant stressed rabbits. Inhibition of protein oxidation was assessed in-vitro by loss of protein thiol (P-SH), while assessment of the reducing power of the extract was further used to assess antioxidant capacity. Results obtained showed the ability of the extract to scavenge free radicals and reactive intermediates in a dose-response manner. The plant also had good antioxidant capacity. The secondary plant metabolites found earlier in the extract may explain reasons for the bio-efficacy of the plant. These findings are of great importance in view of the availability of the plant and its observed possible diverse applications in medicine and nutrition.

Free Radical Scavenging and In-vitro Antioxidant Effects of Ethanol Extract of the Medicinal Herb Chromolaena odorata Linn.

The ethanol extract of the leaf of Chromolaena odorata (Linn) was assessed for free-radical-scavenging and antioxidant potentials. Ability of the extract to scavenge reactive intermediates (superoxide ion O2·-, hydrogen peroxide H2O2, nitric oxide NO˙, hydroxyl radical OH˙) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, were used to assess its free radical scavenging potentials. Antioxidant potential was studied by assessing in-vitro inhibition of lipid peroxidation in both the brain (Neuro-protective potentials) and liver homogenates of Fenton-oxidant stressed rabbits. Inhibition of protein oxidation was assessed in-vitro by loss of protein thiol (P-SH), while assessment of the reducing power of the extract was further used to assess antioxidant capacity. Results obtained showed the ability of the extract to scavenge free radicals and reactive intermediates in a dose-response manner. The plant also had good antioxidant capacity. The secondary plant metabolites found earlier in the extract may explain reasons for the bio-efficacy of the plant. These findings are of great importance in view of the availability of the plant and its observed possible diverse applications in medicine and nutrition.

Mechanisms of γ-hydroxybutyric acid production during the early postmortem period

Although postmortem production of γ-hydroxybutyric acid (GHB) has been confirmed, its production pathways and substrates have not been sufficiently clarified. To confirm that the residual enzymes are involved in GHB production during the early postmortem period, endogenous GHB concentrations in the postmortem blood and liver samples obtained from charred cadavers were compared with those from noncharred cadavers within 50 h after death. The endogenous GHB concentrations in blood and the livers of charred cadavers were significantly lower than those of noncharred cadavers, showing that heat denaturation of enzyme proteins had prevented the postmortem GHB production. In addition, in vitro experiments with rabbit liver homogenates were carried out for inhibition of postmortem GHB production by specific enzyme inhibitors; N-formylglycine (succinic semialdehyde dehydrogenase inhibitor), valproic acid [γ-aminobutyric acid (GABA) aminotransferase inhibitor], pyrazole (alcohol dehydrogenase inhibitor), or N,N-diethyldithiocarbamic acid (aldehyde dehydrogenase inhibitor) was added and incubated, and the inhibition of GHB production was then measured. The results showed that the inhibition ratios by N-formylglycine, valproic acid, and pyrazole were 74.0%, 10.4%, and 17.7%, respectively. This suggests that the contribution ratios of the GHB-producing pathways originating from succinic acid, GABA/putrescine, and 1,4-butanediol are about 70%, 10%, and 20%, respectively.

Biotransformation of Desoxypeganine by Microsomal Enzymes of the Rabbit Liver

The biotransformation of the anticholinergic quinazoline alkaloid Desoxypeganine is studied by means of aerobic incubation with the non-induced supernatant obtained at 9000g from rabbit liver homogenates as enzyme source followed by an admixture of NADPH. The metabolites were identified by high-performance liquid chromatography, chemical ionisation mass spectrometry (LC-CI MS) and electron impact mass spectrometry (LC-EI MS) in comparison with synthetic reference compounds and typical ion fragments taken from literature data. C-oxidation of Desoxypeganine to the major metabolite Pegenone was observed as well as the hydroxylation of the alicyclic ring. The incubation mixture followed Michaelis-Menten kinetics characterised by K(m) = 5.8 x 10(-5) mol L(-1) and V(max) = 4.32 nmol Pegenone/min per mg protein or 3.37 nmol Pegenone/min per nmol CYP 450, respectively. These in vitro results demonstrate that the bioactive substance Desoxypeganine is easily oxidised to its ineffective metabolite Pegenone. This provokes a problem for correct dosage finding in formulations for the treatment of Alzheimer's disease and in the therapy of alcoholism and nicotine dependence.

Bisphosphonate prodrugs: synthesis and in vitro evaluation of novel acyloxyalkyl esters of clodronic acid.

Novel tetra-, tri-, and P,P'-dipivaloyloxymethyl esters of clodronic acid were synthesized, and their properties as possible prodrugs of clodronate were evaluated in vitro. All pivaloyloxymethyl esters were significantly more lipophilic (log P(app) ranged from -2.1 to 7. 4) than clodronate (log P(app) < or = -5.4), which suggests that it may be possible to change the intestinal absorption mechanism of clodronate from a paracellular to a transcellular pathway by a prodrug approach. Pivaloyloxymethyl esters degraded rapidly in 10% rabbit liver homogenate, and half-lives of tri- and P,P'-diesters were 1.1 and 14 min, respectively. The intermediate degradation products were further degraded, and clodronic acid was released in quantitative amounts. In human serum, the stability of pivaloyloxymethyl esters was comparable to their stability in phosphate buffer (pH 7.4), which suggests that their degradation in human serum is mostly due to the chemical hydrolysis. Benzoyloxypropyl esters of clodronic acid were also synthesized, but they did not release clodronic acid due to the enzymatic and chemical stability of the formed 3-hydroxypropyl phosphonate esters and are, therefore, not prodrugs.

Studies on the hydrogenation of the progestagen dienogest in vivo and in vitro in the female rabbit

The biotransformation of the progestagen dienogest (17α-cyanomethyl-17β-hydroxy-4,9-estradien-3-one) was studied in vivo in female rabbits and in vitro by liver homogenates from female rabbits and rats. In vivo, in the female rabbit, 3H-dienogest was the subject of an extensive biotransformation. A significant difference between the composition of the urinary and biliary metabolite patterns of dienogest was found. While in the urinary metabolite pattern more polar metabolites dominated, in bile of animals with a bile fistula, a dienogest metabolite of medium polarity was prevalent. This main metabolite of dienogest was identified by MS, 1H- and 13C-NMR spectroscopy and CD measurement of an enzymatic dehydrogenation product as the tetrahydro metabolite 17α-cyanomethyl-5α-estr-9-en-3β,17β-diol. Thus, in vivo, the 4,9-dien-3-oxo-19-norsteroid dienogest is hydrogenated to a 5αH-9-en metabolite. In vitro, however, 3H-dienogest was only poorly transformed by liver homogenates from both species, whereas 3H-levonorgestrel and 3H-3-keto-desogestrel were converted partially by liver homogenates from female rabbits and completely by liver homogenates from female rats. The principal biotransformation reactions of levonorgestrel and 3-ketodesogestrel were the reduction of the 3-oxo group to 3-OH and the 5β- and 5α-hydrogenation of the 4-double bond by homogenates of female rabbit liver and female rat liver, respectively. A dihydro metabolite of dienogest, in which the 3-oxo group had been reduced to 3-OH, was isolated in small amounts from the incubation with rabbit liver homogenate. The data indicate that the enzymatic hydrogenation of the 4-double bond of the 4,9-dien-3-oxo steroid dienogest is inhibited in vitro. The hindered hydrogenation reaction in vitro has to be seen in association with the 9-double bond in the steroid molecule.

Thalidomide prolonged graft survival in a rat cardiac transplant model but had no inhibitory effect on lymphocyte function in vitro

The effects of thalidomide on in vitro interleukin 2 (IL-2) production and thymidine uptake by human peripheral blood lymphocytes or rat splenocytes were investigated. Phytohaemagglutinin-stimulated human lymphocytes were incubated in the presence of thalidomide added at culture initiation. No immunosuppressive effect of thalidomide was observed in these experiments. Primary human mixed lymphocyte cultures treated with thalidomide for 6 days were also unaffected. A microsomal rabbit liver homogenate was prepared for metabolizing thalidomide. Stimulated lymphocytes secreted significantly more IL-2 in the presence of microsomal-treated thalidomide than did controls. The effect of thalidomide was then studied either as single therapy or in combination with cyclosporin A (CyA) in a rat allograft cardiac transplantation model. In addition, T cell subsets were analysed by flow cytometry in untransplanted rats treated with thalidomide. Treatment was given as induction therapy from the day of transplantation until day 9. Graft survival in rats treated with thalidomide was significantly prolonged compared to the untreated group. No difference in graft survival was detected between rats treated with thalidomide or CyA only. Graft survival was found to be slightly prolonged in rats given thalidomide and CyA in combination compared to rats treated with CyA alone. In untransplanted rats given thalidomide a decrease of CD4 positive T cells was detected on days 3 and 5. The T helper/cytotoxic-suppressor cell ratio was significantly diminished but, after 1 week of treatment, values for T cell subsets had almost returned to baseline levels. No inhibitory effect was obtained when phytohaemagglutinin-stimulated rat splenocytes were cultured with metabolized thalidomide. In summary, the ability of thalidomide to improve allograft survival in a solid organ transplant model was verified. The occurrence of thalidomide-induced changes in T cell subset ratios was demonstrated. In in vitro studies, however, there was no decrease but an increase in IL-2 production, and no change in thymidine uptake. The mechanism responsible for the immunosuppressive effect of thalidomide remains to be elucidated.

Rabbit hemorrhagic disease virus (RHDV): ultrastructure and biochemical studies of typical and core-like particles present in liver homogenates

Calicivirus particles isolated from rabbits suffering from acute RHD were compared with virions found in rabbits with chronic disease. Liver homogenates of rabbits with the protracted disease display no hemagglutinating activity and contain viral particles with diameters of 25–27 nm. These virions contain only one structural protein of 30 kDa and are distinctly smaller than intact rabbit hemorrhagic disease virus (RHDV) 32–40 nm). To prove the RHDV identity of the smaller virions, their reactivity with RHDV specific antibodies was investigated by immunoblots of the virion protein and by immunoelectron microscopy. Proteolytic digestion of RHDV particles with α-chymotrypsin did not transform RHDV into the smaller form. We assume that these core-like particles (CLPs) are not a result of proteolytic digestion but arise from a truncated RHDV genome or defective expression.

Diagnosis of rabbit haemorrhagic disease (RHD) and European brown hare syndrome (EBHS) by indirect sandwich polyclonal ELISA.

The establishment of two indirect sandwich polyclonal ELISAs for the virological diagnosis of rabbit haemorrhagic disease (RHD) and European brown hare syndrome (EBHS) is described. Each assay uses rabbit and guinea pig antisera raised to RHD or EBHS purified virus particles. The tests are sensitive and specific, but cross-reactions between rabbit haemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV) are observed. Most rabbit liver homogenates identified as RHDV-positive by ELISA do not haemagglutinate human red blood cells at room temperature.

Evaluation of hydroxyl radical-scavenging property of garlic.

Garlic has been reported to provide protection against hypercholesterolemic atherosclerosis and ischemia-reperfusion-induced arrhythmias and infarction. Oxygen free radicals (OFRs) have been implicated as causative factors in these diseases and antioxidants have been shown to be effective against these conditions. The effectiveness of garlic in these disease states could be due to its ability to scavenge OFRs. However, the OFR-scavenging activity of garlic is not known. Also it is not known if its activity is affected by cooking. We therefore investigated, using high pressure liquid chromatography, the ability of garlic extract (heated or unheated) to scavenge exogenously generated hydroxyl radical (.OH). .OH was generated by photolysis of H2O2 (1.2-10 mumoles/ml) with ultraviolet (UV) light and was trapped with salicylic acid (500 nmoles/ml). H2O2 produced .OH in a concentration-dependent manner as estimated by .OH adduct products 2,3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA. Garlic extract (5-100 microliters/ml) produced an inhibition (30-100%) of 2,3-DHBA and 2,5-DHBA generated by photolysis of H2O2 (5.00 pmoles/ml) in a concentration-dependent manner. Its activity is reduced by 10% approximately when heated to 100 degrees C for 20, 40 or 60 min. The extent of reduction in activity was similar for the three heating periods. Garlic extract prevented the .OH-induced formation of malondialdehyde in the rabbit liver homogenate in a concentration-dependent manner. It alone did not affect the MDA levels in the absence of .OH. These results indicate that garlic extract is a powerful scavenger of .OH and that heating reduces its activity slightly.

Purification and partial characterization of an indomethacin hydrolyzing enzyme from pig liver.

Indomethacin is well known to be metabolized via O-demethylation and N-deacylation. In this paper we found an enzyme involved in the hydrolysis of amide-linkage of indomethacin and partially characterized it as well as its substrate specificity. An indomethacin hydrolyzing enzyme was purified to homogeneity from pig liver microsomes using columns of Q-Sepharose, Red-Sepharose and Blue-Sepharose. The enzyme activity was assayed by measuring of rho-chlorobenzoic acid liberated from indomethacin by HPLC. The purified enzyme effectively hydrolyzed the amide linkage in indomethacin but not those in alpha-naphthylacetate and rho-nitrophenylacetate, which are typical substates for carboxylesterase. The subunit molecular mass of the enzyme was 65 kDa according SDS-polyacrylamide gel electrophoresis. The Michaelis constant (K(m)) and maximum velocity (Vmax) values for indomethacin were 67.8 microM and 9.02 nmol/min/mg protein, respectively. The amino acid sequence analysis of the enzyme after cyanogen bromide cleavage showed high homology with a mouse carboxylesterase isozyme designated as ES-male. The activity of indomethacin hydrolysis was relatively high in the pig, rabbit and human liver homogenate, but not in those from rat and mouse. On the other hand, purified human liver carboxylesterases pl 5.3 and 4.5, and pig liver carboxylesterases have no catalytic activity for indomethacin. These results indicate that the hydrolysis of amide-linkage of indomethacin in humans would be associated with an enzyme similar to the indomethacin hydrolyzing enzyme from pig liver microsomes described here.

Iron Overload Augments the Development of Atherosclerotic Lesions in Rabbits

Iron, a major oxidant in vivo, could be involved in atherosclerosis through the induction of the formation of oxidized LDL, a major atherogenic factor. This study was designed to test this hypothesis experimentally. Four groups of New Zealand White rabbits were included: iron-overloaded/hypercholesterolemic (group A, n = 8), iron-overloaded (group B, n = 6), hypercholesterolemic (group C, n = 6), and untreated (group D, n = 6). Iron overload was achieved by the intramuscular administration of 1.5 g of iron dextran divided in 30 doses. Hypercholesterolemia was produced by feeding rabbit chow enriched with 0.5% (wt/wt) cholesterol. Serum iron, ferritin, cholesterol, triglycerides, and lipoperoxides in serum were measured throughout the study. Lipoperoxides were measured at the end of the study in liver, aorta, and spleen homogenates. Aortas of groups A and C had multiple lesions; however, group A had greater lesional involvement than group C (P < .05). Lesions were not observed in rabbits fed normal chow (group D). As expected, serum iron and ferritin were above normal levels in groups A and B. Serum cholesterol increased in groups A and C. Lipoperoxides in liver and spleen homogenates of iron-overloaded rabbits were increased. Interestingly, iron deposits were seen by ultrastructural studies in the arterial walls of rabbits in groups A and B. Our study suggests that iron overload augments the formation of atherosclerotic lesions in hypercholesterolemic rabbits.

N-Alkoxycarbonyl prodrugs of mebendazole with increased water solubility

Bioreversible derivatization of mebendazole to improve its poor water solubility, which is attributed to its low peroral bioavailability, was performed by N-acylation of the benzimidazole moiety with various chloroformates. The N-alkoxycarbonyl derivatives were readily hydrolyzed to mebendazole in rabbit liver homogenate and in human and rabbit plasma. The pH-rate profiles for the hydrolysis of the derivatives were derived at 37°C, and the lipophilicity of the compounds was assessed by partition experiments in octanol-aqueous buffer systems. The water solubility of the N-alkoxycarbonyl derivatives was up to 16-times higher than that of the parent drug. The results obtained suggest that N-alkoxycarbonyl derivatives of benzimidazole anthelmintics may be useful to improve the peroral bioavailability.

Prodrugs of peptides. 18. Synthesis and evaluation of various esters of desmopressin (dDAVP).

Various aliphatic carboxylic acid esters and a carbonate ester of the tyrosine phenolic group in desmopressin were synthesized to assess their suitability as prodrugs with improved bioavailability compared to the parent peptide. The chemical stability of the esters in aqueous solution was similar to that of simple phenol esters. The derivatives were quantitatively converted to desmopressin by enzymatic hydrolysis in human plasma and rabbit liver homogenate. The esters with a straight side chain were rapidly hydrolyzed by alpha-chymotrypsin, but the sterically hindered pivalate ester proved more stable than desmopressin itself toward this proteolytic enzyme. All the esters were more lipophilic than desmopressin in terms of octanol-buffer partition coefficients. The transport of the compounds across confluent monolayers of Caco-2 cells was examined. No correlation between permeability and lipophilicity was found but the pivalate ester showed a markedly higher flux relative to desmopressin. It is concluded that appropriate esterification of desmopressin at its tyrosine group may be a potentially useful prodrug approach.

Identification and characterization of an acyl-CoA:triterpene acyltransferase activity in rabbit and human tissues.

Rabbit and human tissues contain substantial amounts of an unusual lipid, a fatty acid ester of a pentacyclic triterpene, that is a potent in vitro inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT). A possible origin of the triterpene ester is via dietary absorption of plant triterpenes (which have a similar structure to the triterpene moiety of the animal triterpene ester), followed by fatty acid esterification of the triterpene in animal tissues. To support this idea, homogenates of rabbit and human enterocytes and liver are now shown to contain an acyl-CoA:triterpene acyltransferase activity (ATAT) which esterifies triterpene to a fatty acid. The enzyme activity was stimulated by exogenous triterpene and required ATP and coenzyme A when fatty acid was used as substrate; ATP and coenzyme A were not required when fatty acyl-CoA was used. ATAT was not inhibited by two structurally different ACAT inhibitors, which may indicate that ACAT and ATAT are different enzymes. Rat enterocytes and liver contained very little ATAT activity, consistent with the finding that rat liver contained very little triterpene ester. To establish that triterpene esterification occurs in vivo, [3H]triterpene was shown to be incorporated into triterpene ester in several organs and tissues from a rabbit given a gastric bolus of the labeled triterpene. These data provide support for the hypothesis that triterpene esters in animal tissues arise from the dietary absorption of triterpenes followed by the esterification of the triterpenes by an enzymatic activity in the animal tissues.

A study of growth regulators of renal cortical tubular cells in the rabbit liver

Two growth regulators, a growth stimulator for cultured renal tubular cells and a growth inhibitor, were observed in this study in rabbit liver homogenate after a unilateral nephrectomy. These regulators appeared in the liver on the third day after this nephrectomy, decreased on the seventh day, and disappeared by the fourteenth day. The growth stimulatory activity, termed a tubular cell growth factor (TuCGF), was a heat- and acid stable 15 to 20 KDa protein. The additive effects of TuCGF on the epidermal growth factor (EGF), the insulin-like growth factor-I (IGF-I), and the fibroblast growth factor (FGF) were observed; the results suggest that TuCGF differs from EGF, IGF-I, or FGF. In contrast, the growth inhibitory activity, termed a tubular cell growth inhibitor (TuCGI), was a heat- and acid-labile protein with a molecular weight of about 150 to 200 KDa. This factor potently inhibited the DNA synthesis of tubular cells in the presence of insulin and EGF. These results suggest that TuCGI is not identical with transforming growth factor-beta (TGF-beta). The regulation of these activities by target cell density is also discussed.