Impaired DNA Synthesis Research Articles

Inhibition of osteoblastic cell proliferation and ornithine decarboxylase activity by ethanol.

Low bone mass and an increased prevalence of skeletal fractures are evident in the alcoholic population. Histomorphometric analysis of skeletal tissue from alcoholic patients reveals reduced osteoblast number and suppressed bone formation activity, with relative sparing of resorptive indexes. The decreased number of osteoblasts observed in alcoholic subjects results from either impaired proliferation or accelerated senescence. Polyamines and ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine synthesis, are essential for cell proliferation in a variety of cell types. To determine whether the consequences of ethanol on osteoblast number involve the modulation of polyamine biosynthesis, we examined the effect of ethanol on parameters of cell growth and ODC activity in a rat osteoblast-like osteosarcoma cell line (UMR 106-01). Ethanol markedly impaired DNA synthesis and cell proliferation in a dose-dependent fashion. Difluoromethylornithine, a specific inhibitor of ODC activity, induced a similar inhibition of UMR 106-01 cell proliferation, indicating the importance of the polyamine pathway in this osteoblastic cell line. Induction of ODC activity was impaired in ethanol-exposed cell cultures in a dose-dependent fashion that paralleled the antiproliferative effects. Finally, supplemental polyamine administration substantially improved DNA synthesis in ethanol-exposed UMR 106-01 cell cultures. These data confirm a direct inhibitory effect of ethanol on osteoblast proliferation that may in part explain the reduced bone mass observed in subjects who consume excessive amounts of alcohol. These findings also suggest that altered polyamine metabolism may be an important mechanism responsible for the antiproliferative effects of ethanol on the osteoblast.

The mechanism of recA polA lethality: suppression by RecA-independent recombination repair activated by the lexA(Def) mutation in Escherichia coli.

The mechanism of recA polA lethality in Escherichia coli has been studied. Complementation tests have indicated that both the 5'-->3' exonuclease and the polymerization activities of DNA polymerase I are essential for viability in the absence of RecA protein, whereas the viability and DNA replication of DNA polymerase I-defective cells depend on the recombinase activity of RecA. An alkaline sucrose gradient sedimentation analysis has indicated that RecA has only a minor role in Okazaki fragment processing. Double-strand break repair is proposed for the major role of RecA in the absence of DNA polymerase I. The lexA(Def)::Tn5 mutation has previously been shown to suppress the temperature-sensitive growth of recA200(Ts) polA25::spc mutants. The lexA(Def) mutation can alleviate impaired DNA synthesis in the recA200(Ts) polA25::spc mutant cells at the restrictive temperature. recF+ is essential for this suppression pathway. recJ and recQ mutations have minor but significant adverse effects on the suppression. The recA200(Ts) allele in the recA200(Ts) polA25::spc lexA(Def) mutant can be replaced by delta recA, indicating that the lexA(Def)-induced suppression is RecA independent. lexA(Def) reduces the sensitivity of delta recA polA25::spc cells to UV damage by approximately 10(4)-fold. lexA(Def) also restores P1 transduction proficiency to the delta recA polA25::spc mutant to a level that is 7.3% of the recA+ wild type. These results suggest that lexA(Def) activates a RecA-independent, RecF-dependent recombination repair pathway that suppresses the defect in DNA replication in recA polA double mutants.

Apoptosis mediates and thymidine prevents erythroblast destruction in folate deficiency anemia.

Deficiency of the vitamin folic acid causes pancytopenia by decreasing the production of new blood cells. Although impaired DNA synthesis and destruction of hematopoietic cells have been implicated, the mechanism by which folate deficiency decreases blood cell production is uncertain. An in vitro model of folate-deficient erythropoiesis was developed by using proerythroblasts isolated from folate-deficient mice that were infected with Friend leukemia virus. Proerythroblasts from folate-deficient mice had one-tenth the total folate as did proerythroblasts from control mice. The folate-deficient proerythroblasts underwent apoptosis, a form of programmed cell death, after 20-32 h in culture in folate-deficient medium. At the time of apoptosis the cells had differentiated into the later erythroblast stages and some had begun hemoglobin synthesis. Addition of either folic acid or thymidine, but not deoxycytidine or inosine, to the folate-deficient medium prevented the apoptosis and permitted proliferation and differentiation of the proerythroblasts into reticulocytes. The prevention of apoptosis by thymidine indicates (i) that decreased thymidylate synthesis plays a role in erythroblast apoptosis and the anemia of folate deficiency and (ii) that DNA cleavage is likely to be a primary event in the apoptosis of folate-deficient erythroblasts. Apoptosis of erythroblasts in the late stages of differentiation leads to decreased erythrocyte production and to anemia. The increased erythropoietin produced in response to the anemia increases the number of erythroid progenitor cells in the differentiation stages preceding those in which the cells undergo apoptosis. This population shift to earlier stage erythroblasts and proerythroblasts is characteristic of bone marrows of individuals with folate deficiency anemia.

Assembly of the Bacteriophage T4 Replication Machine Requires the Acidic Carboxy Terminus of Gene 32 Protein

The acidic carboxy-terminal 89-amino acid fragment of bacteriophage T4 gene 32 protein was expressed in Escherichia coli to high levels from an inducible plasmid construct. Infection of induced cells by wild-type T4 phage results in impaired phage DNA synthesis. The time at which DNA synthesis begins and the diminution in DNA synthesis rates correlate with the amount of carboxy-terminal peptide that accumulates intracellularly prior to infection. Correspondingly, when induced cells are induced cells are infected with viable phage containing a small deletion near the carboxy-terminus of 32 protein (ΔPR201), the inhibition of phage DNA synthesis was much more severe. The mutant 32 protein competes less well against overproduced wild-type acid peptide than does wild-type 32 protein. The purified acid peptide, when used as the attached ligand for affinity chromatography, binds several T4 proteins from phage-infected cells, including 43 protein (T4 DNA polymerase), Dda protein (a DNA helicase), and UvsX protein (a Rec-like recombination protein). Furthermore, at 50- to 100-fold molar excess of acid peptide over intact 32 protein, phage DNA synthesis was specifically inhibited at the initiation step in an in vitro 5-protein DNA replication experiment. We propose that one or more phage replication proteins are titrated as non-productive protein-protein complexes at a site away from the DNA template. This implies that the carboxy-terminal domain of 32 protein is involved in an obligate step of replication machine assembly when the protein is properly attached to ssDNA in the vicinity of a primer-template junction. The assembly defect we observe is strikingly similar to the repression, or "squelching", of the activity of certain eukaryotic transcriptional activators.

Copper deficiency reversibly impairs DNA synthesis in activated T lymphocytes by limiting interleukin 2 activity.

The essentiality of adequate copper (Cu) nutriture for normal T-cell function in laboratory and domestic animals is well established. However, specific biochemical roles of Cu in the maturation and activation of T cells have not been defined. Previous work showed that when cultures of splenic mononuclear cells (MNCs) from Cu-deficient rats were exposed to T-cell mitogens, DNA synthesis was markedly reduced despite normal up-regulation of interleukin 2 (IL-2) receptors, transferrin receptors, and class II major histocompatibility complex molecules. In the present study, IL-2 activity in PHA-treated cultures of MNCs from Cu-deficient rats was 40-50% that of controls as determined by bioassay. Addition of rat IL-2 to phytohemagglutinin-treated cultures of MNCs from Cu-deficient rats increased blastogenic activity to control levels, demonstrating that Cu deficiency does not inhibit transition of quiescent cells to the competence phase of the activation process. Moreover, supplementation of MNC cultures from Cu-deficient rats with physiological levels of Cu enhanced IL-2 activity and DNA synthesis in response to phytohemagglutinin. These data indicate that IL-2 activity in cultures of activated splenic T lymphocytes from Cu-deficient rats is insufficient for optimal blastogenesis.

The genotoxicity of the anti-cancer drug mitoxantrone in somatic and germ cells of Drosophila melanogaster

The novel antineoplastic drug mitoxantrone was studied for its genotoxic effects in Drosophila melanogaster. In male germ cells, the clinical preparation Novantrone ®, the dihydrochloride salt of mitoxantrone, did not induce sex-linked recessive lethal mutations in feeding and injection experiments with adult flies, although statistically the results were inconclusive rather than truly negative. However, the free base mitoxantrone was weakly, but significantly genotoxic in this test (0.14% lethals/mM exposure concentration); this is most probably the result of prolonged exposure. On the other hand, both forms of mitoxantrone assayed were clearly genotoxic in the somatic mutation and recombination test of the wing. This test assays the cells of the proliferating imaginal wing discs of larvae. Depending on the feeding method used, the overall clone induction frequency was in the range of about 2–6 × 10 −5 per cell and cell generation and per mM exposure dose. Correction of these frequencies according to mean clone size led to slightly higher estimates (by about 5–25% higher). Although the majority of the clone induction events are due to mitotic recombination, a significant proportion can be attributed to mutational events (gene and chromosome mutations). The genotoxicity of mitoxantrone seems to depend mainly on impaired DNA synthesis in cycling cells owing to the compound's ability to inhibit topoisomerase II by intercalation into DNA.

Modification of radiation induced genetic damage and impaired DNA synthesis by thiourea treatment in Solanum incanum L.

Modification of induced genetic damage after exposure to LD50 and LD90 doses of 60Co gamma-irradiation on dormant seeds of Solanum incanum L. by pre- and post-treatments of thiourea was investigated. Thiourea pre-treatment reduced cellular lesions, growth injury and the death of seedlings, while post-treatment increased lethality. Incorporation of 3H-tymidine into DNA fraction gradually increased with 10-4 to 10-2 M thiourea treatment when applied before irradiation. Post-treatment of the thiourea, on the other hand, not only showed poor labelling of DNA but also delayed its synthesis.

Effect of marginal zinc nutriture on protein and lipid metabolism in rat pancreas in vitro

To determine the effect of marginal zinc nutriture on the pancreas, rats were divided into three groups and fed Rodent Blox ad libitum, 4-ppm-zinc-supplemented liquid diet ad libitum, and 50-ppm-zinc-supplemented liquid diet in amounts isocaloric to the 4-ppm-zinc supplemented diet, for a period of 15 wk. Compared to Rodent Blox-fed animals, animals fed diet supplemented with 4 ppm zinc and with 50 ppm zinc had lower body weights and higher pancreas weights in relation to body weight. DNA synthesis and total content and specific activity of amylase were decreased, whereas trypsinogen, chymotrypsinogen, and lipase were not affected in animals fed diet supplemented with 4 ppm zinc (compared with animals fed 50-ppm-zinc-supplemented diet). Lipid studies revealed increased free and esterified cholesterol and a decreased level of triglyceride. Incorporation of14C-1 -acetate in triglyceride was increased. Electron microscopy showed no change in number, size, and volume fraction of zymogen granules/unit. These studies indicate that marginal zinc nutriture alters the function of the pancreatic acinar cell, independent of the caloric intake in the diet. Although marginal zinc nutriture was expected to impair DNA synthesis, isolated decrease of amylase seems to be mediated through altered insulin and/or glucose homeostasis. Changes observed in lipid metabolism may underlie the membrane pathology associated with zinc deficiency.

Synergy of inhibition of DNA synthesis in human bone marrow by azidothymidine plus deficiency of folate and/or vitamin B12?

The effect of azidothymidine (Zidovudine, AZT) on pyrimidine (thymidine, deoxyuridine, and thymidine triphosphate) incorporation into DNA in folate- and/or vitamin B12-deficient and normal human bone marrow cells was studied to investigate whether such vitamin deficiency affects susceptibility to AZT-induced hematologic toxicity. Bone marrow cells from 12 patients were studied: 5 had folate and/or vitamin B12 deficiency; 7 controls included 5 with anemia related to chronic disease and 2 with iron deficiency. At 0.2 microM AZT (3 hr, 37 degrees C), the approximate pharmacologic serum trough level, pyrimidine incorporation into DNA was suppressed by 12 to 19% in folate- and/or vitamin B12-deficient cells and by 16 to 23% in normal cells. At 2.0 microM AZT (3 hr, 37 degrees C), the approximate pharmacologic serum peak level, this was suppressed by 15 to 40% in folate- and/or vitamin B12-deficient cells and by 32 to 47% in controls. Deoxyuridine incorporation into DNA was inhibited significantly greater than thymidine at 2.0 microM AZT (3 hr, 37 degrees C) in both groups. Inhibition of deoxyuridine incorporation was not reversed with methyltetrahydrofolate or vitamin B12. There tended to be less striking suppression by AZT of deoxyuridine incorporation into DNA in bone marrow cells from vitamin B12-deficient patients, which was made more striking by adding vitamin B12. This suggests that some of what passes for "AZT damage" to bone marrow cells may in fact be coincident deficiency of vitamin B12. AZT inhibition of DNA synthesis in 3 hr bone marrow cultures is relatively consistent in a variety of hematologic disorders. As approximately two-thirds of AIDS patients appear to be in negative balance with respect to folate and/or vitamin B12, the fact that AZT-induced inhibition of pyrimidine incorporation into DNA is occurring in cells which may be megaloblastic, i.e., in a state of impaired DNA synthesis, suggests that these cells may be more susceptible to AZT toxicity. The data also support the notion that AZT inhibition results predominantly from termination of DNA chain elongation. Whether folate or vitamin B12 supplementation may partially overcome apparent "AZT inhibition" of DNA synthesis (hematologic toxicity) and whether the benefit of such therapy exceeds the risk will require further study.

2'-Deoxycytidine protects normal human bone marrow progenitor cells in vitro against the cytotoxicity of 3'-azido-3'-deoxythymidine with preservation of antiretroviral activity

Bone marrow cytotoxicity of 3′-azido-3′-deoxythymidine (AZT), an anti- human immunodeficiency virus (anti-HIV) drug, has been attributed to deoxyribonucleotide pool perturbations that might result in impaired DNA synthesis in normal bone marrow elements. We examined, in vitro, the effect of high, but clinically achievable and nontoxic, concentrations of 2′-deoxycytidine (dCyd) (greater than or equal to 100 mumol/L) on high-dose AZT mediated growth inhibition and intracellular biochemical perturbations in normal bone marrow progenitor cells. Colony formation by bone marrow progenitor cells in semisolid medium was significantly protected by dCyd against the inhibitory effects of co-administered, high concentrations of AZT (10 mumol/L). Also, dCyd significantly corrected AZT mediated depletion of intracellular thymidine triphosphate (dTTP) and dCyd triphosphate (dCTP) levels in normal bone marrow mononuclear cells (BMMC). Moreover, dCyd reduced the intracellular accumulation of AZT triphosphate (AZT-TP) and its DNA incorporation in BMMC. In contrast, co-administration of dCyd (100 mumol/L to 1 mmol/L) did not reverse AZT (10 mumol/L) mediated suppression of HIV infectivity in HUT-102 cells in culture, although a partial reduction in intracellular AZT-TP pools and its DNA incorporation as well as a correction of AZT mediated depletion of dTTP and dCTP pools was observed in these cells. These studies suggest that dCyd at high concentrations might ameliorate the bone marrow cytotoxicity of high-dose AZT without impairing its anti-HIV effect.

2’-Deoxycytidine Protects Normal Human Bone Marrow Progenitor Cells In Vitro Against the Cytotoxicity of 3′-Azido-3′-Deoxythymidine With Preservation of Antiretroviral Activity

Bone marrow cytotoxicity of 3'-azido-3'-deoxythymidine (AZT), an anti-human immunodeficiency virus (anti-HIV) drug, has been attributed to deoxyribonucleotide pool perturbations that might result in impaired DNA synthesis in normal bone marrow elements. We examined, in vitro, the effect of high, but clinically achievable and nontoxic, concentrations of 2'-deoxycytidine (dCyd) (greater than or equal to 100 mumol/L) on high-dose AZT mediated growth inhibition and intracellular biochemical perturbations in normal bone marrow progenitor cells. Colony formation by bone marrow progenitor cells in semisolid medium was significantly protected by dCyd against the inhibitory effects of co-administered, high concentrations of AZT (10 mumol/L). Also, dCyd significantly corrected AZT mediated depletion of intracellular thymidine triphosphate (dTTP) and dCyd triphosphate (dCTP) levels in normal bone marrow mononuclear cells (BMMC). Moreover, dCyd reduced the intracellular accumulation of AZT triphosphate (AZT-TP) and its DNA incorporation in BMMC. In contrast, co-administration of dCyd (100 mumol/L to 1 mmol/L) did not reverse AZT (10 mumol/L) mediated suppression of HIV infectivity in HUT-102 cells in culture, although a partial reduction in intracellular AZT-TP pools and its DNA incorporation as well as a correction of AZT mediated depletion of dTTP and dCTP pools was observed in these cells. These studies suggest that dCyd at high concentrations might ameliorate the bone marrow cytotoxicity of high-dose AZT without impairing its anti-HIV effect.

Pituitary dependence of bone marrow function

The anaemia, leucopenia, thrombocytopenia and impaired DNA and RNA synthesis in the bone marrow of hypophysectomized rats could be restored by syngeneic pituitary grafts placed under the kidney capsule, or by treatments with ovine or bovine prolactin or growth hormone. Treatment with ACTH, FSH, LH and TSH had no effect in this respect. These results indicate that bone marrow function is regulated by the pituitary gland.

Impaired Dna Synthesis by Early Pubertal Cryptorchid Testis In Vitro

To study the spermatogenic potential of the early pubertal cryptorchid testis, testicular DNA synthesis was examined in vitro. DNA synthesis by early pubertal cryptorchid testis was inhibited and lost its normal temperature sensitivity. The spermatogenic potential of the cryptorchid testis may be impaired as early as puberty.

Different mechanisms account for the suppression of interleukin 2 production and the suppression of interleukin 2 receptor expression in Trypanosoma brucei‐infected mice

Lymph node cell populations derived from Trypanosoma brucei-infected mice failed to produce interleukin 2 (IL 2) in response to a potent mitogenic trigger and suppress the potential of normal lymph node cells to secrete IL 2 in co-culture assays. This suppression is promptly restored by the addition of indomethacin, which blocks prostaglandin synthesis, but is not markedly affected by the addition of catalase, which degrades H2O2. The suppression of the IL 2 receptor expression, on the other hand, is not restored by the addition of indomethacin, nor by the simultaneous supply of both indomethacin and catalase. This discrepancy is not caused by an extreme susceptibility of the receptor expression to low prostaglandin (PG) concentrations, but rather by the presence of suppressive cells that operate through a PG-independent mechanism. This suppressive mechanism accounts for the loss of the IL 2 receptors on both the Ly-2+ and the L3T4+ T cell compartment. The indomethacin-treated co-cultures, which manifest a normal IL 2 production but lack the IL 2 receptors, manifest an impaired DNA synthesis and contain a decreased number of T cell blasts.

Autoradiographic Study of the Effects of Psoralen and Ultraviolet Light on DNA, RNA and Protein Synthesis in Cultured Keratinocytes and Psoriatic Skin

DNA, RNA and protein synthesis were studied by autoradiography in cultured keratinocytes, immediately, 24 and 48 h after 8-methoxypsoralen and ultraviolet light, (PUVA) treatment. Using the same technique, the immediate and long-term effects of PUVA therapy on DNA, RNA and protein synthesis were analysed in skin biopsies from psoriatic patients. In the cultures, immediately after irradiation, DNA and RNA syntheses were similarly inhibited in a dose-dependent manner, while protein synthesis was slightly affected only for the highest dose. After 24 h, RNA synthesis recovered whereas DNA synthesis was more severely inhibited suggesting that other cell components may be damaged by PUVA. In patients, DNA and RNA syntheses decreased immediately after PUVA sessions. During all the sessions until the psoriatic plaques had cleared, an impairment of DNA synthesis was observed in comparison with the synthesis in involved and uninvolved skin before treatment. These results suggest that the therapeutic efficiency of PUVA is based on the inhibition of DNA replication due to direct effects on nucleic acids but also to photoreactions with other cell components.

Studies of erythroblast function in congenital dyserythropoietic anaemia, type I: evidence of impaired DNA, RNA, and protein synthesis and unbalanced globin chain synthesis in ultrastructurally abnormal cells.

Two patients with congenital dyserythropoietic anaemia, type I (CDA) were studied. Their blood reticulocytes showed unbalanced globin chain synthesis with increased alpha:beta globin chain synthesis ratios. A high proportion of the erythroblasts displayed the characteristic "Swiss cheese" abnormality of the nuclear chromatin and some also showed cytoplasmic intrusions lined with nuclear membrane within the nucleus. Occasional erythroblast profiles contained intracytoplasmic inclusions that were ultrastructurally indistinguishable from precipitated alpha chains. The technique of combined Feulgen microspectrophotometry and 3H-thymidine autoradiography showed gross abnormalities of proliferation in the early polychromatic erythroblasts. The proliferative abnormalities included an arrest of DNA synthesis after the progress of cells through part of the S phase and the formation of several mononucleate and binucleate cells with hypertetraploid total DNA contents. The bone marrow cells gave a normal deoxyuridine suppressed value, indicating that there was no impairment of the methylation of deoxyuridylate. Electron microscope autoradiographic studies showed that a high proportion of the erythroblasts with the "Swiss cheese" nuclear abnormality suffered from a severe impairment, or arrest of DNA, RNA, and protein synthesis.

Dissociation of methionine synthetase (EC 2.1.1.13) activity and impairment of DNA synthesis in fruit bats (Rousettus aegyptiacus) with nitrous oxide-induced vitamin B12 deficiency.

1. The effect of methylcobalamin inactivation by the anaesthetic gas nitrous oxide on the activity of the cobalamin-dependent methionine synthetase (5-methyltetrahydrofolate homocysteine methyltransferase; EC 2.1.1.13) reaction, and on DNA synthesis, in the fruit bat Rousettus aegyptiacus, was examined. 2. Methionine synthetase activity in the liver of bats exposed to N2O-oxygen (50:50, v/v) for 90 min/d averaged 32% of that of controls after 4 d of exposure and only 5% after 12-14 weeks of exposure. 3. DNA synthesis in the bone marrow, as reflected by the deoxyuridine suppression test, was unaffected by 4 d of exposure to N2O and only minimally affected after 5-10 weeks of exposure. 4. These results suggest that DNA synthesis in the fruit bat is unusually resistant to inhibition of methionine synthetase and imply the existence of a non-methylated circulating folate pool in this species.

Enhancement of methotrexate cytotoxicity by uracil analogues that inhibit deoxyuridine triphosphate nucleotidohydrolase (dUTPase) activity.

Impairment of DNA synthesis by lack of thymine nucleotides was long considered the critical cytotoxic event in cells exposed to methotrexate (MTX) (1). Recent work has shown, however, that the thymineless state is associated with increases in intracellular dUMP and dUTP with resulting uracil misincorporation into DNA (2-4). Subsequent unrepaired excisions of uracils by uracil-DNA-glycosylase (5,6) lead to DNA fragmentation (7-11).

Inhibitory effects of anti-angiogenic agents on neovascularization and growth of the chorioallantoic membrane (CAM). — The possibility of a new CAM assay for angiogenesis inhibition

Protamine sulfate and combinations of heparin-cortisone acetate known as having anti-angiogenic activities impaired the growth of chorioallantois at the dose inducing no decrease in growth rate of the embryos. This inhibitory effect of the agents is presumed to be mediated by the specific inhibition of the growth of endothelial cells forming chorioallantoic blood vessels based on the following results: significant correlations were found between the length of CAM vessels measured by an automatic image analyzer and the estimated volumes of chorioallantois (correlation coefficient r = 0.94) and these agents specifically inhibited the (3H)-thymidine incorporation into cultured endothelial cells at the dose having no effects on that into cultured chick embryonic cells. On the other hand, all DNA-synthesis inhibitors, mitomycin C, 5-fluorouracil, and paraformaldehyde suppressed the growth of both CAM and embryo and resulted in early embryonic death, which might be due to the nonspecific impairment of DNA synthesis by these agents. These results indicate the possibility that the present CAM assay could screen agents having anti-angiogenic activity.

Restriction of gut-derived endotoxin impairs DNA synthesis for liver regeneration.

The influence of restricting gut-derived endotoxin availability on liver regeneration after partial hepatectomy was evaluated. Partial hepatectomy was performed by 67% liver resection of ether-anesthetized rats. Liver regeneration was quantified after partial hepatectomy by [3H]thymidine incorporation into hepatic DNA; endotoxemia due to absorption of endogenous endotoxin from the gut into the portal circulation was determined by qualitative lysate assay of perchloric acid-extracted plasma samples, and plasma levels of the hepatotrophic factors insulin and glucagon were measured by radioimmunoassay. Treatments to restrict gut-derived endotoxin included chronic gavage with neomycin and cefazolin for gut sterilization, chronic gavage with cholestyramine to bind endotoxin within the gut, subcutaneous administration of polymyxin B to neutralize the lipid A portion of circulating endotoxin, intraperitoneal induction of endotoxin tolerance by progressively higher doses of endotoxin, and experimentation with isolator-reared defined flora Fisher rats that were Gram-negative bacteria deficient and therefore endotoxin deficient. All treatments to restrict endogenous endotoxin impaired DNA synthesis in regenerating livers particularly 21 h posthepatectomy when replication was increasing most rapidly in normal rats. We hypothesize that impairment of DNA synthesis after partial hepatectomy in endotoxin-restricted animals was due to the observed lack of normal systemic endotoxemic as well as hyperinsulinemic and hyperglucagonemic responses to 67% liver resection.