Testing in vitro of an indirect mutagen (cyclophosphamide) with human leukocyte cultures: Activation by liver perfusion and by incubation with crude liver homogenate.

The indirect mutagen cyclophosphamide was tested in human whole blood cultures with respect to chromosome-breaking activities and suppression of mitotic indices after activation by liver perfusion and crude liver homogenates with and without cofactors. Both methods produced nearly the same effects, with the exception of liver homogenate without cofactors which had only weak metabolic activities.     

Characterization of succinate dehydrogenase and alpha-glycerophosphate dehydrogenase in pancreatic islets

Succinate dehydrogenase activities in homogenates of rat and ob/ob mouse pancreatic islets were only 13% of the activities in homogenates of liver and were also several times lower than in homogenates of pancreatic acinar tissue. This indicates that the content of mitochondria in pancreatic islet cells is very low. The very low activity of succinate dehydrogenase is in agreement with the low mitochondrial volume in the cytoplasmic ground substance of pancreatic islet cells as observed in morphometric studies. This may represent the poor equipment of pancreatic islet cells with electron transport chains and thus provide a regulatory role for the generation of reducing equivalents and chemical energy for the regulation of insulin secretion. The activities of succinate dehydrogenase in tissue homogenates of pancreatic islets, pancreatic acinar tissue, and liver were significantly inhibited by malonate and diazoxide but not by glucose, mannoheptulose, streptozotocin, or verapamil. Tolbutamide inhibited only pancreatic islet succinate dehydrogenase significantly, providing evidence for a different behavior of pancreatic islet cell mitochondria. Therefore diazoxide and tolbutamide may affect pancreatic islet function through their effects on succinate dehydrogenase activity. The activities of alpha-glycerophosphate dehydrogenase in homogenates of pancreatic islets and liver from rats and ob/ob mice were in the same range, while activities in homogenates of pancreatic acinar tissue were lower. None of the test agents affected alpha-glycerophosphate dehydrogenase activity. Thus the results provide no support for the recent contention that alpha-glycerophosphate dehydrogenase activity may be critical for the regulation of insulin secretion.     

In vivo induction of sister-chromatid exchanges in liver and marrow cells by drugs requiring metabolic activation

A highly sensitive method for the detection of in vivo induction of sister-chromatid exchange (SCE) has been developed in mice subjected to partial hepatectomy. SCE induction by either acetylaminofluorene (AAF) or cyclophosphamide, drugs requiring metabolic activation, is significantly greater in both regenerating liver and bone-marrow cells of partial hepatectomized animals than in marrow cells of unhepatectomized mice. These experiments have confirmed the ability of AAF, a well known mutagen-carcinogen, to induce SCE formation, even though the cytogenic effects of this drug on non-hepatectomized mice is very small. The in vivo system described has demonstrated the influence of the liver on drug-induced damage to extra-hepatic tissues. The procedures developed should facilitate the detection of drug-induced cytogenic damage and permit the comparison of inter-tissue differences in SCE induction with tissue-specific differences in drug-activation pathways.     

Specific changes in the protein composition of rat liver in response to the peroxisome proliferators ciprofibrate, Wy-14,643 and di-(2-ethylhexyl)phthalate.

The hypolipidaemic agents ciprofibrate and Wy-14,643 ([4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid) and the phthalate-ester plasticizer di-(2-ethylhexyl)-phthalate (DEHP), like other peroxisome proliferators, produce a significant hepatomegaly and induce the peroxisomal fatty acid beta-oxidation enzyme system together with profound proliferation of peroxisomes in hepatic parenchymal cells. Changes in the profile of liver proteins in rats following induction of peroxisome proliferation by ciprofibrate, Wy-14,643 and DEHP have been analysed by high-resolution two-dimensional gel electrophoresis. The proteins of whole liver homogenates from normal and peroxisome-proliferator-treated rats were separated by two-dimensional gel electrophoresis using isoelectric focusing for acidic proteins and nonequilibrium pH gradient electrophoresis for basic proteins. In the whole liver homogenates, the quantities of six proteins in acidic gels and six proteins in the basic gels increased following induction of peroxisome proliferation. Peroxisome proliferator administration caused a repression of three acidic proteins in the liver homogenates. By the immunoblot method using polyspecific antiserum against soluble peroxisomal proteins and monospecific antiserum against peroxisome proliferation associated Mr 80000 polypeptide (polypeptide PPA-80), the majority of basic proteins induced by these peroxisome proliferators appeared to be peroxisomal proteins. Polypeptide PPA-80 becomes the most abundant protein in the total liver homogenates of peroxisome-proliferator-treated rats. These results indicate that ciprofibrate, DEHP and Wy-14,643 induce marked changes in the profile of specific hepatic proteins and that some of these changes should serve as a baseline to identify a set of gene products that may assist in defining the specific 'peroxisome proliferator domain'.     

Influence of metabolic factors on the mutagenic effectiveness of cyclophosphamide in Drosophila melanogaster

This paper describes the influence of changes in metabolic activity on the in-vivo mutagenic effectiveness of cyclophosphamide in Drosophila melanogaster. A dose-dependent increase in mutagenicity was observed until a plateau value is reached which was increased only slightly after enzyme induction with Aroclor 1254, whereas induction with phenobarbital resulted in a decrease, especially when cyclophosphamide was applied by injection. Treatment of the adult males with inhibitors of the monoamine oxidase (MAO, EC 1.4.3.4), such as iproniazid (Ipr), benzimidazole or tryptamine, led to a marked increase of the mutagenic effectiveness of cyclophosphamide especially in spermatocytes. This indicates the importance of metabolic de-activation processes for the limited mutagenicity of cyclophosphamide in Drosophila. The principal active metabolite of cyclophosphamide, phosphoramide mustard, is extensively de-activated by enzymes that can be inhibited by 1-phenylimidazole (PhI), presumably cytochrome P-450 (EC 1.14.14.1), but not by those blocked by MAO inhibitors. Inhibition of the FAD-containing dimethylaniline monooxygenase (FDMAM, EC 1.14.13.8) by N,N-dimethylbenzylamine (N,N-DMB) resulted in some increase in cyclophosphamide mutagenicity only in spermatids. The marginal mutagenicity of cyclophosphamide in Drosophila larvae could not be increased either by cytochrome P-450 induction with phenobarbital or by MAO inhibition with Ipr. In contrast to the failure of cyclophosphamide to induce rod-chromosome loss, a considerable activity was found when a ring-shaped chromosome was used. Similar to the sex-linked recessive lethal (SLRL) test, ring-X loss frequency could be enhanced by simultaneous treatment with MAO inhibitors. The observed ring-X loss frequency declined when males treated with cyclophosphamide were mated to DNA-repair deficient mei-9L1 females. Cyclophosphamide produces chromosome breaks, detected as 2-3 translocations, in Drosophila spermatocytes, the stage in spermatogenesis that is also the most sensitive to the induction of SLRL mutations.     

Short-term tests for transplacentally active carcinogens. A comparison of sister-chromatid exchange and the micronucleus test in mouse foetal liver erythroblasts

The effectiveness of 6 chemicals (benzo[a]pyrene, (BaP), cyclophosphamide (CP), diethylnitrosamine (DEN), methyl methanesulphonate (MMS), mitomycin C (MC) and procarbazine (PC) ) as inducers of micronuclei in foetal liver and maternal bone marrow erythroblasts has been determined, and related to that of gamma-radiation. CP, DEN, MMS and PC were all more effective in the foetal liver. The induction of micronuclei and SCEs by each chemical in foetal erythroblasts after in vivo exposure was measured. When expressed as induction of sister-chromatid exchanges (SCEs) per erythroblast/induction of micronuclei per erythroblast (/microM/kg), the ratios obtained were MC 580, BaP 470, DEN 430, CP 258, MMS 140 and PC 13. The lowest doses detected as potentially genotoxic by each test in foetal liver erythroblasts are (with the exception of PC which is a relatively ineffective inducer of SCEs) similar. When isolated foetal livers were exposed in vitro, SCE dose responses to BaP, MC, MMS and PC could be directly related to those from in vivo exposure, indicating the role of the foetal liver in metabolic activation, but CP was considerably more cytotoxic. The transplacental micronucleus test, and in vivo/in vitro method for SCEs in foetal liver erythroblasts, provide sensitive, complementary assays for genotoxic effects of chemicals during prenatal life. Since foetal liver possesses greater metabolic potential than adult bone marrow, the transplacental tests respond to genotoxic agents not detected by bone-marrow systems.     

Lipid peroxidation measured as thiobarbituric acid-reactive substances in tissue slices: characterization and comparison with homogenates and microsomes

Liver slices were used to measure lipid peroxidation induced by bromotrichloromethane, tert-butyl hydroperoxide (t-BOOH), or ferrous iron. The responses of liver homogenates and microsomes to oxidative conditions were compared with the response of tissue slices. Lipid peroxidation was evaluated by the production of thiobarbituric acid-reactive substances (TBARS). As was observed in homogenates and microsomes, TBARS production by liver slices depended upon the amount of tissue, the incubation time, inducer, the amount of inducer, and the presence of antioxidant. Control liver slices incubated at 37 degrees C for 2 h produced 19 nmol of TBARS per g of liver. When slices were incubated in the presence of 1 mM BrCCl3, 1 mM t-BOOH, or 50 microM ferrous iron, TBARS production increased 4.6-, 8.2-, or 6.7-fold over the control value, respectively. Comparable induction of TBARS by liver homogenates and microsomes was observed when these preparations were incubated with the same inducers. Addition of 5 microM butylated hydroxytoluene (BHT) prevented the induction of TBARS by 50 microM ferrous iron by liver slices. The results indicate the usefulness of tissue slices to measure lipid peroxidation. The usefulness of tissue slices is emphasized when a number of compounds or tissues are studied and tissue integrity is desired as in toxicological, pharmacological, and nutritional studies where reduced numbers of experimental animals is a relevant issue.     

Identification of Monohydroxy metabolites of 15,16-dihydrocyclopenta(a)phenanthren-17-one and its carcinogenic 11-methyl homolog produced by rat liver preparations in vitro.

Incubation of 15,16-dihydrocyclopenta[a]phenanthren-17-one and its carcinogenic 11-methyl homolog with rat liver microsomes led to similar patterns of metabolites. The carcinogen was the more slowly metabolized, but both ketones gave the corresponding 15-hydroxy derivatives, together with small quantities of the isomeric 16-ols. The 11-hydroxymethyl-17-ketone also occurred as a minor carcinogen metabolite. Incubation of the carcinogen with rat liver homogenates caused more extensive metabolism. The ratio of mono-ols to more polar metabolites was similar with homogenates from untreated and methylcholanthrene-induced rats, but increased metabolism to polar derivatives was observed after phenobarbitone induction.     

Effect of peroxisome proliferation on ether phospholipid biosynthesizing enzymes in rat liver

1. The effect of the peroxisome proliferators clofibrate and plasticizer on the activities of the first two enzymes involved in either phospholipid biosynthesis, i.e. dihydroxyacetone-phosphate acyltransferase (DHAP-AT) and alkyldihydroxyacetone-phosphate synthase, were studied in rat liver homogenates and purified peroxisomes. 2. DHAP-AT in homogenates increased by 2 to 3-fold both in total and specific activity. However, the specific activity in purified peroxisomes showed no significant increase demonstrating for the first time that there is no specific induction of this enzyme that exceeds the induction of total peroxisomal protein. 3. Alkyldihydroxyacetone-phosphate synthase showed no significant increase in total and specific activity in homogenates and a slight decrease of its specific activity in purified peroxisomes was observed. 4. The total amount of plasmalogens did not increase upon proliferation and a slight decrease in the percentage plasmalogens in total phospholipids was observed. 5. Proliferation did not influence the phospholipid composition of the peroxisomal membrane.     

Induction of specific-locus and dominant-lethal mutations by cyclophosphamide and combined cyclophosphamide-radiation treatment in male mice

Cyclophosphamide is the most widely used antineoplastic agent. It is also used to condition patients for bone-marrow transplantations. Because of the general interest of this compound we initiated a systematic study of the induction of dominant-lethal and specific-locus mutations in male mice. In addition, we investigated the induction of specific-locus mutations by the combined treatment of cyclophosphamide and ionizing radiation.A dose of 40 mg/kg bw of cyclophosphamide caused dominant-lethal mutations in male mice only in the 1st and 2nd week after treatment. A dose of 120 mg/kg induced dominant-lethal mutations in the mating intervals 1–21 days posttreatment. No dominant lethal mutations were observed after the 3rd week. The same differential spermatogenic response was observed for the induction of specific-locus mutations. Cyclophosphamide induced recessive mutations exclusively in spermatozoa and spermatids. No mutations were recovered from treated spermatocytes and spermatogonia. In contrast to cyclophosphamide, radiation induces specific-locus mutations in all germ-cell stages.The pretreatment with cyclophosphamide 24 h before radiation enhanced the frequency of specific-locus mutations in spermatogonia. The distribution of the observed mutations among the 7 loci and their viability supports the hypothesis that these mutations were induced by radiation rather than by cyclophosphamide. The compound causes an immediate inhibition of DNA and RNA synthesis in spermatogonia. The inhibition very likely interferes with the repair process. The disturbance of the repair process is probably the cause of the synergistic effect for the induction of specific-locus mutations in spermatogonia of mice after pretreatment with cyclophosphamide 24 h before irradiation.     

Copper-metallothionein induction in the liver of LEC rats.

Recently, copper (Cu) was found to be unusually accumulated, suggesting the induction of metallothionein (MT) in the liver of LEC rats (Long-Evans rats with a cinnamon-like coat color), which develop spontaneous jaundice with hereditary hepatitis. Thus, the direct relationship between the unusual Cu accumulation and the induction of Cu-MT was investigated by giving LEC rats Cu-overloaded or Cu-deficient diets. Results based on the determinations of Cu and MT levels in several organs, as well as the gel-filtration profiles of the cytosols of liver homogenates, showed that dietary Cu induced Cu-MT and development of hepatic injury associated with jaundice.     

Differential enzyme induction of mouse liver and lung following a single low or high dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

The induction response of cytochrome P-450-dependent enzyme activities to a single low (5 nmol/kg) or high (50 nmol/kg, intraperitoneal [ip] dose of TCDD was examined in liver and lung homogenates over a 12-week time course in an outbred, Ah-responsive strain of mice (National Institutes of Health [NIH] Swiss). Total hepatic cytochrome P-450 was quantified, and the dealkylation of ethoxy- and benzyloxyresorufin (activities of P-450 IA1 and IIB1, respectively) were measured in both tissues at 48 and 96 hr and at 1, 4, and 12 weeks post-TCDD administration. Western immunoblotting with monoclonal antibody 1-7-1 was conducted to confirm the specific IA1-inductive effects of each dose of TCDD over the same time course. Following the low dose, specific IA1 induction was apparent in liver at the earliest time point, was maximal at 1 week, and declined to control values at 12 weeks. Pulmonary IA1 was near-maximally induced at 48 hr, and remained at that level for 4 weeks. In contrast, a tenfold higher dose of TCDD elicited similar IA1 induction profiles for both tissues, with a maximum at 1 week and a progressive loss at 4 and 12 weeks postexposure. P-450 IIB1 activity was elevated in TCDD-treated animals by enzymatic assay; however, Western immunoblotting did not confirm this finding. These data demonstrate persistent dose-dependent P450 induction over many weeks by a single TCDD dose, with significant organ-specific differences: (a) lung is more sensitive than liver to a nonmaximal inducing dose of TCDD, and (b) at a maximally inducing dose of TCDD, lung is very similar to liver in both the level and time course of IA1 induction.     

Purification and Immunological Characterization of Mouse Hepatic Phosphoenolpyruvate Carboxykinase

The inhibition of glucocorticoid induction and tryptophan activation of phosphoenolpyruvate carboxyfcinase (PEPCK) by bacterial endotoxin may be explained either by decreased synthesis or by inactivation of the enzyme. To differentiate between the two possibilities, mouse hepatic PEPCK was purified using a modification of a method used for securing the enzyme from livers of other species. The techniques included high speed centrifugation of whole liver homogenates, ammonium sulfate fractionation, Sephadex G-100 filtration, DEAE-cellulose ion exchange, hydroxylapatite chromatography, and isoelectric focusing. Antibodies prepared against the purified enzyme gave a single precipitin line in gel diffusion and in Immunoelectrophoresis. The antibodies were used for enzyme titrations in whole liver homogenates of normal and endotoxin poisoned mice, as reported elsewhere.     

肝再生增强因子的cDNA克隆、表达及表达产物的生物活性研究

以肝部分切除后再生肝组织为起始材料,利用RT-PCR扩增出大鼠肝再生增强因子（ALR）,亚克隆于pGEM-T载体,核苷酸序列测定证实为大鼠ALR;将ALRcDNA亚克隆于pBV220质粒,构建了原核表达栽体,并获高效表达菌株,特异表达蛋白占细菌总蛋白的15％,原核表达的ALR在体外缺乏促进大鼠原代培养肝细胞及SMMC-7721肝癌细胞DNA合成的活性,但在体内1／3肝部分切除模型中可刺激肝细胞DNA合成;ALR在生物学活性方面与肝脏刺激物（HSS）存在一定差别,ALR和HSS应是两种不同的活性因子．ALR还具有促肝损伤修复的作用,对其深入研究可能为临床治疗严重肝病提供有效的药物.     

Endogenous factor in rat liver that converts tyrosine aminotransferase form III to form I: a rapid assay of this converting factor.

After induction by cortisol, tyrosine aminotransferase (l-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) present in rat liver homogenates can be resolved into three peaks of activity by CM-Sephadex chromatography. Based on differential elution of these forms by a linear KCl gradient, a three-tube assay was developed that quantitates the amount of form III relative to total enzyme. The assay was used to determine the presence of a factor in the liver that converts tyrosine aminotransferase form III to form I. Definitive evidence for the liberation of such a factor is presented.     

In vitro anti-oxidant property of protein-A of Staphylococcus aureus.

Protein-A (PA) is a cell-surface glycoprotein of S. aureus Cowan I with immunomodulatory and anti-tumor activities, and ability to ameliorate cyclophosphamide and carbontetrachloride (CCl4) induced toxicity in rodents. The likely mechanism of this effect appears to be the anti-oxidant property of PA, evidenced in the present study by inhibition of CCl4 and Fe2-ascorbate induced lipid peroxidation in rat liver homogenates and inhibition of deaminative-oxidative degradation of L-glutamate into 2-thiobarbituric acid reactive products in a constituted chemical system. The anti-oxidant property of PA seem to arise from its molecular characteristics and the ability to interact with a superoxide derived free-radical species without any affinity for superoxide anion, hydroxyl radical and singlet oxygen species.     

A comparative study of some more important experimental animal peroxide metabolism enzymes.

1. We have studied and compared the peroxide metabolism enzymes (SOD, P and C) of the main organs of fresh-water mollusc, chicken, mouse, guinea-pig, rabbit, cat and dog. 2. The liver exhibited the highest SOD activity. The enzymatic activities of the organ homogenates of the guinea-pig stand out in comparison with the values for the other homogenates examined. 3. The liver, kidney and total brain homogenates of the chicken and the vertebrates display no, or only a very low P activity. The highest P activities were measured in the haemolysates. 4. The C and SOD values exhibit a certain parallelism. 5. The peroxide metabolism enzyme activities calculated by utilizing the protein measurements permit the establishment of a more realistic enzymatic activity.     

Induction of hepatic microsomal gamma-glutamyltransferase activity following chronic alcohol consumption.

Chronic alcohol consumption for 4–5weeks results in an enhancement of serum gamma-glutamyltransferase activity in rats. Concomitantly, an increase of hepatic gamma-glutamyltransferase was observed. Upon subcellular fractionation of liver homogenates by ultracentrifugation, an induction of gamma-glutamyltransferase activity could be demonstrated in the microsomal fraction of the hepatocyte. These findings suggest that increased serum gamma-glutamyltransferase activities commonly observed in alcoholism can be ascribed at least in part to an induction of microsomal gamma-glutamyltransferase activity.     

Epoxy derivatives of aromatic polycyclic hydrocarbons. The preparation and metabolism of epoxides related to benzo[a]pyrene and to 7,8- and 9,10-dihydrobenzo[a]pyrene

Products that appeared to be mainly benzo[a]pyrene 7,8-oxide and benzo[a]pyrene 9,10-oxide were synthesized and their chemical and biochemical properties were investigated. The oxides were unstable and readily rearranged to phenols. They were converted by rat liver homogenates and microsomal preparations into phenols and dihydrodiols, but glutathione conjugates were not formed in appreciable amounts. The dihydrodiols formed from benzo[a]pyrene 7,8- and 9,10-oxide by rat liver microsomal preparations were identical in their chromatographic and spectrographic properties with dihydrodiols formed when benzo[a]pyrene was metabolized by rat liver homogenates. 9,10-Dihydrobenzo[a]pyrene 7,8-oxide and 7,8-dihydrobenzo[a]pyrene 9,10-oxide were also synthesized. They were converted by rat liver homogenates and microsomal preparations into the related cis- and trans-dihydroxy compounds. Glutathione conjugates were formed from the oxides by rat liver homogenates. Both 7,8- and 9,10-dihydrobenzo[a]pyrene were metabolized by rat liver homogenates to mainly the trans-isomers of the related dihydroxy compounds. In experiments with boiled homogenates, the benzo[a]pyrene oxides were converted into phenols, whereas the dihydrobenzo[a]pyrene oxides yielded small amounts of the related dihydroxy compounds.     

Hydrolysis of retinyl palmitate by enzymes of rat pancreas and liver. Differentiation of bile salt-dependent and bile salt-independent, neutral retinyl ester hydrolases in rat liver

Previous studies have demonstrated that homogenates of the livers of rats contain a neutral retinyl ester hydrolase activity that requires millimolar concentrations of bile salts for maximal in vitro activity. The enzymatic properties of this neutral, bile salt-dependent retinyl ester hydrolase activity in liver homogenates are nearly identical to those observed in the present report for the in vitro hydrolysis of retinyl palmitate by purified rat pancreatic cholesteryl ester hydrolase (EC 3.1.1.13). Moreover, anti-rat pancreatic cholesteryl ester hydrolase IgG completely inhibits the bile salt-dependent retinyl ester hydrolase activity of rat liver homogenates whereas normal rabbit IgG does not. We also show that liver homogenates contain a neutral, bile salt-independent retinyl ester hydrolase activity that differs from the bile salt-dependent activity in that 1) its absolute activity does not vary markedly among individual rats, 2) it is not inhibited by antibodies to pancreatic cholesteryl ester hydrolase, and 3) it is localized in the microsomal fraction of liver homogenates. Subfractionation of microsomes demonstrates that the neutral, bile salt-independent retinyl ester hydrolase activity is associated with liver cell plasma membranes and thus may play a role in the hydrolysis of retinyl esters delivered to the liver by chylomicron remnants.