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D L Eaton

Publications and source records attributed to D L Eaton.

At least 37 records · Page 2Linked to original sources
PubMed1998

The efficacy of recombinant thrombopoietin in murine and nonhuman primate models for radiation-induced myelosuppression and stem cell transplantation.

Radiation-induced pancytopenia proved to be a suitable model system in mice and rhesus monkeys for studying thrombopoietin (TPO) target cell range and efficacy. TPO was highly effective in rhesus monkeys exposed to the mid-lethal dose of 5 Gy (300 kV x-rays) TBI, a model in which it alleviated thrombocytopenia, promoted red cell reconstitution, accelerated reconstitution of immature CD34+ bone marrow cells, and potentiated the response to growth factors such as GM-CSF and G-CSF. In contrast to the results in the 5 Gy TBI model, TPO was ineffective following transplantation of limited numbers of autologous bone marrow or highly purified stem cells in monkeys conditioned with 8 Gy TBI. In the 5 Gy model, a single dose of TPO augmented by GM-CSF 24 h after TBI was effective in preventing thrombocytopenia. The strong erythropoietic stimulation may result in iron depletion, and TPO treatment should be accompanied by monitoring of iron status. This preclinical evaluation thus identified TPO as a potential major therapeutic agent for counteracting radiation-induced pancytopenia and demonstrated pronounced stimulatory effects on the reconstitution of immature CD34+ hemopoietic cells with multilineage potential. The latter observation explains the potentiation of the hematopoietic responses to G-CSF and GM-CSF when administered concomitantly. It also predicts the effective use of TPO to accelerate reconstitution of immature hematopoietic cells as well as possible synergistic effects in vivo with various other growth factors acting on immature stem cells and their direct lineage-committed progeny. The finding that a single dose of TPO might be sufficient for a clinically significant response emphasizes its potency and is of practical relevance. The heterogeneity of the TPO response encountered in the various models used for evaluation points to multiple mechanisms operating on the TPO response and heterogeneity of its target cells. Mechanistic mouse studies made apparent that the response of multilineage cells shortly after TBI to a single administration of TPO is quantitatively more important for optimal efficacy than the lineage-restricted response obtained at later intervals after TBI and emphasized the importance of a relatively high dose of TPO to overcome initial c-mpl-mediated clearance. Further elucidation of mechanisms determining efficacy might very well result in a further improvement, e.g., following transplantation of limited numbers of stem cells. Adverse effects of TPO administration to myelosuppressed or stem cell transplanted experimental animals were not observed.

Animals↗
PubMed1998

IL-3 does not contribute to platelet production in c-Mpl-deficient mice.

Thrombopoietin is a lineage-dominant cytokine involved primarily in the control of platelet production. The physiological importance of thrombopoietin (TPO) in the regulation of megakaryocyte and platelet production was demonstrated by the production of mice deficient in TPO or its receptor, c-Mpl. Even though these mice are profoundly thrombocytopenic they maintain a basal level of approximately 10% of the normal count of fully functional platelets. These platelets prevent any abnormal bleeding episodes and highlight the potential importance of other factors in the control of platelet production. Among the factors with in vitro megakaryocytopoietic activity, the most potent is undoubtedly interleukin 3 (IL-3). To analyze the contribution of IL-3 to platelet formation in the absence of TPO, we have generated mice deficient in both c-Mpl and IL-3Ralpha by taking advantage of a natural mutation present in this gene in the A/J mouse. Surprisingly, these double knockout mice did not show any further reduction in their platelet or megakaryocyte counts when compared with c-Mpl-deficient mice. Similarly, progenitors from other lineages that are also reduced in c-Mpl-deficient mice are not further affected by the absence of a functional IL-3Ralpha gene. These results demonstrate that IL-3 alone is not responsible for the production of a basal level of normal platelets in the absence of thrombopoietin signaling.

Animals↗
PubMed1998

The efficacy of recombinant TPO in murine And nonhuman primate models for myelosuppression and stem cell transplantation.

Radiation-induced pancytopenia proved to be a suitable model system in mice and rhesus monkeys to study thrombopoietin (TPO) target cell range and efficacy. TPO was highly effective in rhesus monkeys exposed to the midlethal dose of 5-Gy (300 kV x-rays) TBI, a model in which it alleviated thrombocytopenia, promoted red cell reconstitution, accelerated reconstitution of immature CD34+ bone marrow (BM) cells and potentiated the response to growth factors such as GM-CSF and G-CSF. The accelerated reconstitution of BM CD34+ cells appeared to be reflected by a similar rise in peripheral blood CD34+ cells, both being augmented by concomitant GM-CSF. However, TPO was ineffective following transplantation of limited numbers of autologous BM or highly purified stem cells in monkeys conditioned with 8-Gy TBI. In the 5-Gy model, a single dose of TPO 24 h after TBI was effective in preventing thrombocytopenia and was augmented by GM-CSF. The strong erythropoietic stimulation may result in iron depletion and TPO treatment should be accompanied by monitoring of iron status. In mice, similar observations were made and the importance of dose and dose schedule for stimulation of multilineage repopulating cells versus the lineage-dominant thrombopoietic response studied in detail.

Animals↗
PubMed1998

Metabolism and toxicity of aflatoxins M1 and B1 in human-derived in vitro systems.

Aflatoxin M1 (AFM1) is the principal hydroxylated aflatoxin metabolite present in the milk of dairy cows fed a diet contaminated with aflatoxin B1, (AFB1) and the metabolite is also present in the milk of human nursing mothers consuming foodstuffs containing the toxin. AFM1 is usually considered to be a detoxification product of AFB1 and this appears warranted if the biological endpoints involved are carcinogenicity and mutagenicity. However, it may not be a valid conclusion in the case of cytotoxicity. The metabolism of AFM1 and AFB1 have been studied in vitro using human liver microsomes. Formation of primary metabolites associated with metabolic activation to the respective epoxides reflected the differences between the carcinogenic potentials of the two toxins and, similar to AFB1, the conjugation of AFM1 epoxide with reduced GSH was catalyzed by mouse, but not human liver cytosol. Although the majority of the binding of [3H]AFB1 to microsomal protein was dependent on metabolic activation, a high level of retention of [3H]AFM1 by microsomes, nonextractable in methanol and unrelated to metabolic activation, was observed. It appears possible that this property is related to the high cytotoxicity of AFM1. Experiments using human cell line cells either expressing or not expressing human cytochrome P450 enzymes in assays of acute toxicity (MTT assays) have demonstrated a directly toxic potential of AFM1 in the absence of metabolic activation, in contrast to AFB1. Caution therefore needs to be exercised in designating the formation of AFM1 as essentially detoxification when considering a biological response in which cytotoxicity may play a significant role, e.g., immunotoxicity.

Aflatoxin B1↗
PubMed1998

Identification of amino acid residues essential for high aflatoxin B1-8,9-epoxide conjugation activity in alpha class glutathione S-transferases through site-directed mutagenesis.

Mice constitutively express glutathione S-transferase mGSTA3-3 in liver. This isoform possesses uniquely high conjugating activity toward aflatoxin B1-8,9-epoxide (AFBO), thereby protecting mice from aflatoxin B1-induced hepatocarcinogenicity. In contrast, rats constitutively express a closely related GST isoenzyme, rGSTA3-3, with low AFBO activity and, therefore, are sensitive to aflatoxin B1 exposure. Although the two GSTs share 86% sequence identity and have similar catalytic activities toward 1-chloro-2,4-dinitrobenzene (CDNB), they have an approximately 1000-fold difference in catalytic activity toward AFBO. To identify amino acids that confer high activity toward AFBO, non-conserved rGSTA3-3 residues were replaced with mGSTA3-3 residues in two regions believed to form the substrate binding site. Twenty-one mutant rGSTA3-3 enzymes were generated by site-directed mutagenesis using combinations of nine different residues. Except for the E208D mutant, single mutations of rGSTA3-3 produced enzymes with no detectable AFBO activity. Generally, AFBO conjugation activity increased in additive fashion as mGSTA3-3 residues were introduced into the rGSTA3-3 enzyme with the six site mutant E104I/H108Y/Y111H/L207F/E208D/V217K displaying the highest AFBO activity (40 nmol/mg/min) of all the mutant enzymes. When this mutant enzyme was further modified by three additional substitutions (D103E/I105M/V106I) AFBO conjugation activity decreased 14-fold to 2. 8 nmol/mg/min. Although wild-type mGSTA3-3 AFBO conjugation activity (265 nmol/mg/min) could not be obtained by our rGSTA3-3 mutants, we were able to identify six mGSTA3-3 residues; Ile104, Tyr108, His111, Phe207, Asp208, and Lys217 that, when collectively substituted into rGSTA3-3, substantially increased (>200-fold) glutathione conjugation activity toward AFBO.

Aflatoxin B1↗
PubMed1998

Role of CYP1A2 in the hepatotoxicity of acetaminophen: investigations using Cyp1a2 null mice.

Acetaminophen (APAP) is known to cause centrilobular hepatic necrosis under overdose conditions. This is thought to be mediated via the P450-generated reactive intermediate N-acetyl-p-benzoquinone imine (NAPQI). Initially, NAPQI is detoxified by conjugation with glutathione (GSH), but once GSH is depleted, NAPQI reacts more extensively with hepatic proteins leading to hepatocellular damage. The P450 isoforms thought to be responsible for APAP hepatotoxicity in humans are CYP2E1, CYP1A2, and CYP3A4, and thus, we have investigated the effect of murine Cyp1a2 on APAP hepatotoxicity using Cyp1a2 knockout mice (Liang et al., Proc. Natl. Acad. Sci. USA 93, 1671-1676, 1996). Doses of 250 mg/kg were markedly hepatotoxic in these mice, and surprisingly, deaths only occurred in the knock-out and heterozygote mice over a 24-h period after dosing. Furthermore, there were no significant differences among survivors of any genotype in serum ALT concentrations, a well correlated indicator of APAP hepatotoxicity in mice. Finally, no differences were observed in the urinary metabolites excreted ove the 24-h period, including those derived from GSH conjugation of the major reactive metabolite NAPQI. Consistent with the effects on hepatotoxicity and metabolism, 2 h after hepatotoxic doses (500 mg/kg, i.p.) of APAP no significant differences were observed in total whole liver homogenate nonprotein thiol concentrations among the three genotypes even though hepatic thiols were decreased compared to control animals (> 90%). In addition, when the liver cytosol and microsome samples were examined by immunoblotting for the presence of APAP-protein adducts using a specific antiserum, there were no observable differences in either the intensity of staining or in the spectrum of adducts formed between APAP-dosed mice of any genotype. The cumulative data suggest that Cyp1a2 doses not play a significant role in APAP hepatotoxicity in these mice.

Acetaminophen↗
PubMed1997

The efficacy of single-dose administration of thrombopoietin with coadministration of either granulocyte/macrophage or granulocyte colony-stimulating factor in myelosuppressed rhesus monkeys.

Thrombopoietin (TPO) was evaluated for efficacy in a placebo-controlled study in rhesus monkeys with concurrent administration of either granulocyte/macrophage colony-stimulating factor (GM-CSF) or granulocyte CSF, (G-CSF). Rhesus monkeys were subjected to 5 Gy total-body irradiation (TBI), resulting in 3 weeks of profound pancytopenia, and received either TPO 5 microg/kg intravenously (I.V.) at day 1 (n = 4), GM-CSF 25 microg/kg subcutaneously (S.C.) for 14 days (n = 4), TPO and GM-CSF (n = 4), G-CSF 10 microg/kg/d S.C. for 14 days (n = 3), TPO and G-CSF (n = 4), or placebo (carrier, n = 4; historical controls, n = 8). Single-dose I.V. treatment with TPO 1 day after TBI effectively counteracted the need for thrombocyte transfusions (provided whenever thrombocyte levels were <40 x 10(9)/L) and accelerated platelet reconstitution to normal levels 2 weeks earlier than placebo controls. TPO/GM-CSF was more effective than single-dose TPO alone in stimulating thrombocyte regeneration, with a less profound nadir and a further accelerated recovery to normal thrombocyte counts, as well as a slight overshoot to supranormal levels of thrombocytes. Monkeys treated with TPO/GM-CSF uniformly did not require thrombocyte transfusions, whereas those treated with GM-CSF alone needed two to three transfusions, similar to the placebo-treated monkeys, which required, on average, three transfusions. Also, reticulocyte production was stimulated by TPO and further augmented in monkeys treated with TPO/GM-CSF. TPO alone did not stimulate neutrophil regeneration, whereas GM-CSF shortened the period of neutrophil counts less than 0.5 x 10(9)/L by approximately 1 week; TPO/GM-CSF treatment elevated the neutrophil nadir, but did not further accelerate recovery to normal values. TPO also augemented the neturophil response to G-CSF, resulting in similar patterns of reconstitution following TPO/G-CSF and TPO/GM-CSF treatment. TPO/GM-CSF resulted in significantly increased reconstitution of CD34+ bone marrow cells and progenitor cells such as GM-CFU and BFU-E. Adverse effects of combining TPO with the CSFs were not observed. It is concluded that (1) a single I.V. administration of TPO is sufficient to prevent severe thrombocytopenia following myelosuppression, (2) TPO/G-CSF and TPO/GM-CSF treatment result in distinct response patterns, with TPO/GM-CSF being superior to TPO/G-CSF in stimulating thrombocyte and erythrocyte recovery while being equivalent in stimulating neutrophil recovery; and (3) TPO significantly improves the performance of CSFs in alleviating severe neutropenia.

Animals↗
PubMed1997

Prevention of thrombocytopenia by thrombopoietin in myelosuppressed rhesus monkeys accompanied by prominent erythropoietic stimulation and iron depletion.

The effectiveness of thrombopoietin (TPO) in alleviating thrombocytopenia was evaluated in a placebo-controlled study involving rhesus monkeys exposed to 5 Gy total-body irradiation (TBI) (300-kV x-rays) to result in 3 weeks of pancytopenia. Supraoptimal treatment with human recombinant TPO (10 microg/kg/d subcutaneously, days 1 to 21 after TBI) was highly effective in preventing thrombocytopenia, with nadirs for thrombocytes, on average, far higher than 100 x 10(9)/L, a greatly accelerated recovery to normal values, and no need for thrombocyte transfusions. TPO appeared to act selectively in that neutrophil regeneration was not influenced but red blood cell lineage recovery was prominently stimulated, with reticulocyte regeneration being initiated 10 days earlier than in placebo-treated animals. The reticulocytosis was followed by a normoblastosis that occurred earlier and was more pronounced than in placebo-treated monkeys. The effect of TPO on the red blood cell lineage was also reflected in a less profound nadir for hemoglobin (Hb) and hematocrit values than in placebo controls. However, this effect was not followed by a rapid recovery to normal values, due to development of a microcytic hypochromic anemia. Iron depletion was demonstrated by measurements of total serum iron and total iron-binding capacity (TIBC) and could be prevented by prophylactic intramuscular (IM) iron before TBI or corrected by IM iron after TPO treatment. Rechallenging with TPO in week 8 after TBI demonstrated a homogenous thrombocyte response similar in magnitude to the initial response, but a greatly diminished reticulocyte response. This demonstrated that the erythropoietic response to TPO administration depends on the hemopoietic state of the animal and may reflect multiple TPO target cells. It is postulated that the extremely rapid erythropoiesis due to TPO treatment in the initial regeneration phase following myelosuppression results in iron depletion by a mechanism similar to that seen following erythropoietin treatment in patients with end-stage renal failure. It is concluded that protracted TPO therapy to counteract thrombocytopenic states may result in iron depletion and that the iron status should be monitored before, during, and after TPO treatment.

Animals↗
PubMed1997

Genetic polymorphisms in human drug metabolic enzymes.

Results obtained from both epidemiologic studies and experimental animal model systems have shown a wide range of phenotypic variation in the ability of individuals to metabolize drugs and environmental chemicals. Several studies have noted correlations between specific metabolic phenotypes and the incidence of disease, suggesting that certain allelic forms of drug metabolic enzymes can render the individual either more sensitive or resistant to the toxic or therapeutic effects of exogenous drugs and chemicals. While some of this variation can be attributed to different environmental exposures, it has become clear that genetic factors also play an important role in determining the response of the individual organism to exogenous agents. Recent advances in molecular biological techniques have begun to allow scientists to correlate observed phenotypic differences with the actual differences in genetic sequence at the gene level. This has allowed a correlation between gene structure and function, thus providing a mechanistic basis to explain the interaction between genetic background and individual response to environmental exposures. Results presented at this symposium discussed how genetic polymorphisms for both Phase I and Phase II metabolic enzymes in the human population modulate the response to environmental toxicants.

Humans↗
PubMed1997

Aflatoxin B1 activation in human lung.

Inhalation exposure to the carcinogen aflatoxin B1 (AFB1) in certain occupations is considerable. Because circumstantial epidemiological evidence suggests that AFB1 inhalation may cause primary lung cancer, we investigated AFB1 activation by human lung microsomes. Microsomes were incubated with [3H]AFB1 (124 microM), and activation to the AFB1-8,9-epoxide was measured as the AFB1-glutathione (AFB1-GSH) conjugate by HPLC. The formation of AFB1-GSH was in the range of 0.05-0.073 fmol/mg protein/min. The role of cytochrome P450 (CYP) 3A in this activation was investigated by oxidation of nifedipine (a prototype substrate for CYP 3A), by immunoinhibition, and by immunoblot analysis. Nifedipine oxidation varied from 0.2 to 19.2 pmol/mg protein/min in microsomes from different subjects, but did not correlate with AFB1 activation. Anti-human polyclonal CYP 3A4 IgG inhibited AFB1 activation. CYP 3A isoforms were immunoestimated to be in the range of 0.01-1.90 pmol/mg protein. Neither CYP 1A2 nor associated activity was detected in the lung microsomes. These data indicate that human lung microsomes activate AFB1 to form the exo-AFB1-8,9-epoxide and that CYP(s) of the 3A subfamily may be responsible for this activity. The relatively low amount of AFB1 activation in human lung compared to that in human liver can be explained by the scarcity of CYP-containing cells in the lung. In situ AFB1 activation and resultant carcinogenic risk are distinctly possible in occupational settings where inhalation of AFB1-contaminated dusts occurs.

Aflatoxin B1↗
PubMed1997

Simultaneous administration of TPO and G-CSF after cytoreductive treatment of rhesus monkeys prevents thrombocytopenia, accelerates platelet and red cell reconstitution, alleviates neutropenia, and promotes the recovery of immature bone marrow cells.

Simultaneous treatment with human thrombopoietin (TPO) and granulocyte colony-stimulating factor (G-CSF) was evaluated in a placebo-controlled rhesus monkey study using 5 Gy total body irradiation (TBI) to induce 3 weeks of pancytopenia. Daily administration of TPO (10 microg/kg/day injected subcutaneously [sc] days 1-21 after TBI) promoted platelet and reticulocyte recovery, resulting in less profound nadirs and a rapid recovery to normal levels. Platelet transfusions were not required in these animals, in contrast to controls, and hemoglobin levels stabilized rapidly. TPO treatment did not influence neutrophil counts. G-CSF (5 microg/kg/day sc days 1-21) stimulated neutrophil regeneration and had no effect on platelet levels. Simultaneous treatment with TPO and G-CSF was as effective as treatment with TPO alone in preventing thrombocytopenia, although with the former regimen platelet levels did not rise to the supranormal levels seen with the latter. Neutrophil recovery was greatly augmented compared with G-CSF treatment alone, resulting in a less profound nadir and a recovery that started much earlier, as did monocyte, CD11b+, CD16+, and CD56+ cell reconstitution. In addition, TPO strongly promoted the recovery of bone marrow cellularity and granulocyte/macrophage and erythroid progenitor cells: The number of bone marrow CD34+ cells was greater by two orders of magnitude in TPO-treated animals than in controls in the second week of treatment, whereas G-CSF by itself had no influence. In the third week after TBI an elevation of LDH1 values was observed in TPO-treated monkeys concurrent with normoblastosis; both of these findings were attributed to rapid erythropoiesis. TPO had no effect on hemostasis parameters. Adverse TPO and/or G-CSF effects were not observed. This study demonstrates that simultaneous TPO and G-CSF treatment after cytoreductive treatment prevents thrombocytopenia, accelerates platelet and red cell reconstitution, alleviates neutropenia, and promotes the recovery of immature bone marrow cells. The effect on CD34+ GM progenitor cells may explain the augmented G-CSF responses in TPO-treated monkeys; it also suggests that TPO may become a key growth factor in the design of treatment regimens to accelerate both immature bone marrow and mature blood cell reconstitution after cytoreductive therapy.

Animals↗
PubMed1997

Lack of efficacy of thrombopoietin and granulocyte colony-stimulating factor after high dose total-body irradiation and autologous stem cell or bone marrow transplantation in rhesus monkeys.

The efficacy of recombinant human thrombopoietin (TPO) and recombinant human granulocyte colony stimulating factor (G-CSF) in stimulating platelet and neutrophil recovery was evaluated in a placebo-controlled study involving transplantation of limited numbers (1-3 x 10(4)/kg) of highly purified autologous stem cells (CD34++/RhLA-DR[dull]) into rhesus monkeys after the animals were subjected to 8 Gy of total body irradiation (TBI) (x-rays). The grafts shortened profound TBI-induced pancytopenia from 5 to 6 weeks to 3 weeks. Daily subcutaneous (sc) injection of TPO (10 microg/kg/day, days 1-21 after TBI) did not stimulate platelet regeneration after transplantation either alone or in combination with G-CSF (5 microg/kg/day sc, days 1-21 after TBI). G-CSF treatment failed to prevent neutropenia in the monkeys and did not stimulate recovery to normal neutrophil levels. Simultaneous administration of TPO and G-CSF did not influence the observed recovery patterns. To test the hypothesis that the limited number of cells transplanted or the subset chosen was responsible for the lack of effectiveness of TPO, three additional monkeys were transplanted with 10(7)/kg unfractionated autologous bone marrow cells. Two of these animals received TPO and the other served as a control. In this setting, as well, TPO treatment did not prevent thrombocytopenia. This study demonstrates that treatment with TPO does not accelerate platelet reconstitution from transplanted stem cells after high-dose TBI. These findings contrast with the rapid TPO-stimulated platelet recovery in myelosuppression induced by 5 Gy of TBI in rhesus monkeys; we conclude from this that the clinical effectiveness of the TPO response depends on the availability of TPO target cells in the first week after TBI, that is, before endogenous TPO levels reach the saturation point. In addition, protracted isolated thrombocytopenia was observed in two G-CSF-treated monkeys, one of which also received TPO. Furthermore, TPO treatment for 7 days in the 6th week after TBI during severe thrombocytopenia in one monkey produced prompt clinical improvement and an increase in platelet counts.

Animals↗
PubMed1996

Binding of the aflatoxin-glutathione conjugate to mouse glutathione S-transferase A3-3 is saturated at only one ligand per dimer.

The binding of two different reaction products (p-nitrobenzyl glutathione and the aflatoxin-glutathione conjugate) to mouse glutathione S-transferase A3-3 (mGSTA3-3) has been measured using equilibrium dialysis and a direct fluorescence quenching technique. As expected, p-nitrobenzyl glutathione was found to bind with a stoichiometry of 2.24 +/- 0.17 mol/mol of dimeric enzyme. However, the much larger aflatoxin-glutathione conjugate, 8, 9-dihydro-8-(S-glutathionyl)-9-hydroxyl-aflatoxin B1 (AFB-GSH), was found to bind with a stoichiometry of 1.12 +/- 0.08 mol/mol of dimeric enzyme. p-Nitrobenzyl glutathione bound mGSTA3-3 with a dissociation constant (Kd) of 59 +/- 17 microM while the aflatoxin-glutathione conjugate bound the enzyme with a Kd of 0.86 +/- 0.19 microM. Glutathione competitively inhibited binding of AFB-GSH to mGSTA3-3 with a Ki of 1.5 mM, suggesting that AFB-GSH was binding to the enzyme active site. Although AFB-GSH bound to mGSTA3-3 with a stoichiometry of 1 mol/mol of dimeric enzyme, AFB-GSH completely inhibited activity toward 1-chloro-2, 4-dinitrobenzene, indicating that AFB-GSH binding to one active site alters affinity for 1-chloro-2,4-dinitrobenzene in the active site of the other subunit. To our knowledge, this is the first report of a glutathione S-transferase reaction product which binds to the enzyme with a stoichiometry of 1 mol/mol of dimer.

Aflatoxin B1↗
PubMed1996

Oltipraz-mediated changes in aflatoxin B(1) biotransformation in rat liver: implications for human chemointervention.

Oltipraz (OPZ) is currently being considered for human use to protect against aflatoxin B1 (AFB)-induced hepatocarcinogenesis based on its proven protective effect in rats. The effectiveness of this treatment presumes that orthologous cytochrome P450 and glutathione S-transferase (GST) isozymes metabolize AFB in humans as they do in rats. In this study, alterations in the expression of multiple forms of cytochrome P450 and GST were evaluated after treatment with OPZ, as well as other known P450 inducers, including 3-methylcholanthrene, pregnenolone-16alpha-carbonitrile, and ciprofibrate. Evidence is presented that the male-specific rat CYP 3A2, an orthologue of human CYP 3A4, may be primarily responsible for AFB activation in rat liver at both high and low AFB substrate concentrations. The CYP 1A2 enzyme does not appear to play a role in AFB activation in rat liver at any substrate concentration, whereas the major human P450 enzyme capable of activating AFB at a low substrate concentration has been identified as CYP 1A2. Surprisingly, we found that the CYP 1A2 steady-state mRNA level and the CYP 1A2-associated methoxyresorufin-O-demethylase activity were induced approximately 3- and 2-fold, respectively, by OPZ in rat liver. However, because CYP 1A2 does not appear to participate in AFB activation, induction of CYP 1A2 may be insignificant for AFB-induced hepatocarcinogenesis in rat models. In the rat, a heterodimeric alpha class GST enzyme containing the Yc2 subunit is the only polypeptide characterized to date in this species with high catalytic activity for the conjugation of activated AFB with glutathione. The GST Yc2 steady-state mRNA level was induced 5-fold by OPZ treatment. This induction was mirrored by significant increases in both the corresponding protein level and AFB-8,9-epoxide-conjugating enzyme activity, which may contribute significantly to protection against AFB-induced carcinogenesis in the rat. Investigations from this and other laboratories have not revealed any evidence for a Yc2-like GST isozyme with high AFB-8,9-epoxide-conjugating activity in human liver. We have also been unable to demonstrate that the two major human alpha class GST isozymes, A1-1 and A2-2, purified from bacteria expressing the corresponding cDNAs, exhibit any significant AFB-8,9-epoxide-conjugating activity. Our results suggest that humans may not be protected to the same extent as rats against AFB-induced hepatocarcinogenesis by treatment with OPZ and that further investigations are needed to establish the usefulness of OPZ for protection against human exposure to AFB.

Aflatoxin B1↗
PubMed1996

Physiological regulation of early and late stages of megakaryocytopoiesis by thrombopoietin.

Thrombopoietin (TPO) has recently been cloned and shown to regulate megakaryocyte and platelet production by activating the cytokine receptor c-mpl. To determine whether TPO is the only ligand for c-mpl and the major regulator of megakaryocytopoiesis, TPO deficient mice were generated by gene targeting. TPO-/- mice have a >80% decrease in their platelets and megakaryocytes but have normal levels of all the other hematopoietic cell types. A gene dosage effect observed in heterozygous mice suggests that the TPO gene is constitutively expressed and that the circulating TPO level is directly regulated by the platelet mass. Bone marrow from TPO-/- mice have decreased numbers of megakaryocyte-committed progenitors as well as lower ploidy in the megakaryocytes that are present. These results demonstrate that TPO alone is the major physiological regulator of both proliferation and differentiation of hematopoietic progenitor cells into mature megakaryocytes but that TPO is not critical to the final step of platelet production.

Animals↗
PubMed1996

In vivo biological effects of various forms of thrombopoietin in a murine model of transient pancytopenia.

Thrombopoietin (TPO) is the natural regulator of platelet production in the bone marrow of mammals. This cytokine also seems to play an important role in the development of the erythroid lineage when recovering from anemic conditions. Here we study the effects of various TPO molecules on the recovery of hematopoietic lineages in a mouse model of pancytopenia. Based on previous animal experimentation and clinical experience with other hematopoietic cytokines, we found that daily dosing with TPO augmented the recovery of both the megakaryocyte and erythroid lineages in a mouse model of pancytopenia. However, further experiments showed that no benefit was gained by using more than a single dose of recombinant murine (rm)TPO(335) given 24 h after the initiation of the myelosuppressive treatment. This response to a single dose of rmTPO(335) is dose-dependent. However, the response was attenuated when a truncated, short half-life TPO molecule (rmTPO[153]) was used. Increasing the half-life of the molecule with 10 kDa polyethylene glycol (PEG) does not improve the response. Only when larger PEG molecules (20 kDa or 40 kDa) are linked to the rmTPO(153) is the response to single doses restored to the level of the full-length molecule. These data suggest that, unlike our experience with other cytokines, the commitment of progenitors to a megakaryocytic cell line is accomplished by a single short exposure to TPO.

Animals↗
PubMed1996

Determination of aflatoxin B1 biotransformation and binding to hepatic macromolecules in human precision liver slices.

Although epidemiological studies suggest that aflatoxin B1 (AFB1) is a human carcinogen, at least in the presence of hepatitis B virus infection, animal studies have demonstrated large differences in species sensitivity to AFB1, and the sensitivity of humans relative to experimental animals remains unclear. The purpose of this study was to determine the profile of AFB1 metabolism and the extent of AFB1 binding to cell macromolecules in human liver slices under experimental conditions that would allow direct comparison to similar endpoints in the rat, a species sensitive to the carcinogenic actions of AFB1. Liver slices were prepared from three individual human liver samples with a Krumdieck tissue slicer and incubated with 0.5 microM [3H]AFB1 for 2 hr. Significant interindividual variations were observed in the rates of oxidative metabolite formation and in specific binding to cell macromolecules. The rates of oxidative metabolism of AFB1 to AFQ1, AFP1, and AFM1 in the three human liver samples were similar to those previously observed in rat liver slices. AFB1-GSH conjugate formation was not detected in any of the human liver samples, and yet specific binding of AFB1 to cell macromolecules was considerably lower in the human liver slices relative to that in rat liver slices. AFB1-DNA binding levels ranged from 3 to 26% of control rat and AFB1-RNA binding levels ranged from 25 to 49% of control rat. The AFB1-protein binding level in the one human sample measured was 20% of that observed for control rat. While these results suggest that humans do not form as much AFBO as the rat, they are also consistent with the hypothesis that humans do not possess GST isozyme(s) with high specific activity toward AFBO. Significant individual differences in AFB1 metabolism and binding between humans suggest the presence of genetic and/or environmental factors that may confer large variability in susceptibility to AFB1.

Adult↗