Erythroid colony formation (CFUe) in fetal liver and adult bone marrow and spleen from the mouse

The methyl cellulose modification of the CFUe technique has been applied to 14 day fetal liver and adult bone marrow and spleen from CBA/CA mice. Optimized doses of fetal calf serum, alpha-thioglycerol, erythropoietin and cell suspensions have been obtained from dose response curves in order to standardize the technique. The slopes of the erythropoietin and cell dose response curves indicate a greater sensitivity by fetal liver to the hormone than bone marrow or spleen. The proportion of cells in the DNA synthesis phase of the cell cycle, as measured by the CFUe technique, has been estimated by administering hydroxyurea. Two hours after the drug was injected, 89% of fetal liver cells, 71% of bone marrow cells and 81% of spleen cells were found to be in the S-phase.     

Transplantation of stromal cells transduced with the human IL3 gene to stimulate hematopoiesis in human fetal bone grafts in non-obese, diabetic-severe combined immunodeficiency mice

The non-obese diabetic-severe combined immunodeficiency (NOD-SCID) mouse is a convenient host for human hematopoietic tissues and cells. Human fetal bone fragments engrafted subcutaneously in NOD-SCID mice sustain human hematopoiesis for several months. MS5 murine bone marrow stromal cells were transfected by electroporation with a plasmid containing the human interleukin-3 gene. As expected, stably transfected hu-IL3-MS5 cells supported human hematopoiesis in vitro more efficiently than MS5 cells. hu-IL3-MS5 cells were then injected intravenously into hu-NOD-SCID mice to test their ability to home to the mouse and/or human bone marrow, and to evaluate the role of hu-IL3 secretion on human hematopoiesis in vivo. hu-IL3 was detected in the mouse serum for up to an observation time of 8 weeks. hu-IL3-MS5 cells engrafted the bone marrow, spleen, liver and lungs of the mice but also the human bone graft. The presence of hu-IL3-MS5 cells in the human bone significantly stimulated local human hematopoiesis. This setting could be used to model the bone marrow homing of intravenously injected stromal cells or stromal cell precursors. The same experimental principle could also be applied in a therapeutic perspective to malignant human bone marrow hematopoiesis.     

Bone marrow and renal injury associated with haloalkene cysteine conjugates in calves

Almost 40 years ago, it was reported that cattle-feed which had been extracted with hot trichloroethylene and then fed to calves produced renal injury and a fatal aplastic anaemia. The toxic factor was subsequently identified as S-(1,2-dichlorovinyl)-L-cysteine (DCVC). These original findings have been confirmed, a single intravenous dose of DCVC at 4 mg/kg, or 0.4 mg/kg intravenously per day administered for 10 days to calves produced aplastic anaemia, and renal injury after a single dose of 4 mg/kg. The toxicity to calves of a number of other haloalkene cysteine conjugates has been examined to ascertain whether, like DCVC, they produce bone marrow and renal injury. Intravenous administration of the N-acetyl cysteine conjugate of DCVC produced renal but not bone marrow injury at a molar equivalent dose to DCVC, indicating that the calf can deacetylate the mercapturic acid and further that sufficient chemical had reached the kidney to be a substrate for the enzyme cysteine conjugate beta-lyase. However, intravenous administration of the alpha-methyl analogue of DCVC, which cannot undergo metabolism via the enzyme cysteine conjugate beta-lyase, was without toxicity at doses about five-fold higher than DCVC. These latter findings provide strong evidence that metabolism of DCVC via the enzyme beta-lyase is necessary for bone marrow and renal injury to occur. The cysteine conjugates of perchloroethylene and hexachloro-1,3-butadiene(HCBD) when given intravenously to calves at molar equivalent doses to DCVC, or above, did not produce either bone marrow or renal injury. In contrast, intravenous administration of the cysteine conjugate of tetrafluoroethylene (TFEC) produced severe renal tubular injury in calves without affecting the bone marrow. In vitro studies with these haloalkene cysteine conjugates showed, like DCVC, that they were good substrates for calf renal cysteine conjugate beta-lyase and toxic to renal cells as judged by their ability to reduce organic anion and cation transport by slices of calf renal cortex and inhibit the renal enzyme glutathione reductase. Calves were also dosed either orally or intravenously with HCBD to assess its toxicity. HCBD at higher molar equivalent doses than DCVC produced mid-zonal necrosis in the liver, renal tubular necrosis but no bone marrow injury in calves. The key findings emerging from these studies are (1) that none of the other cysteine conjugates, at molar equivalent doses to DCVC and above, produce bone marrow injury in calves, (2) TFEC produced only renal injury, suggesting that sufficient of the other conjugates had not reached the kidney for metabolism by beta-lyase to produce cytotoxicity and (3) that HCBD itself is more toxic than its cysteine or mercapturic acid conjugate, suggesting that pharmaco-kinetics and disposition are important factors in determining the toxicity of these conjugates to calves. Further studies are needed to understand the basis for the selective toxicity of DCVC to the bone marrow of calves.     

Accessory atrioventricular pathways with decremental conduction (Mahaim connections). 2 case reports

A recently generated aSmase knock-out mouse line develops a lethal storage disease which mimics the neurovisceral form of Niemann-Pick disease in man. In extension to the previously described neuropathological changes, the purpose of this study was to provide a detailed morphological, particularly ultrastructural analysis of the visceral organs of these animals including spleen, liver, intestine, lung, and kidney along with a sequential histological investigation of the bone marrow. Our results showed a progressive lysosomal storage as indicated by an increasing amount of foam cells in the bone marrow with age, extending to all visceral organs. Most severe storage phenomena were found in the mononuclear-macrophage system, however, parenchymal cells of visceral organs were also markedly involved. The ultrastructural appearance of membrane-bound inclusions displayed a pleomorphic aspect ranging from small vesicular and vesiculo-granular structures to huge lysosomes with membranous material deposited in lamellar or stacked arrays. The obvious similarity to its human counterpart along with an easy availability makes this animal model a valuable tool for further studies of Niemann-Pick disease type A.     

5-fluorouracil/leucovorin-induced inhibition of thymidylate synthase in normal tissues of mouse and man

We evaluated the effects of 5-fluorouracil (5FU) and leucovorin (LV) on thymidylate synthase (TS) in normal rapidly dividing tissues, which may contribute to toxic side-effects of treatment with 5FU and LV. TS levels were determined in biopsies of human liver and colon mucosa and murine bone marrow, liver and intestinal mucosa at several time points after administration of therapeutic doses of 5FU or LV/5FU. In murine liver, after treatment with 100 mg/kg 5FU, TS inhibition was significantly higher than after LV/5FU administration (P < 0.001). A similar trend was observed in human liver tissue. Murine intestinal mucosa had TS levels below the limit of detection after 5FU or LV/5FU treatment. In human colon mucosa samples, administration of 500 mg/m2 5FU resulted in a large extent of TS inhibition but the small number of samples did not allow a time- or 5FU-LV/5FU-related evaluation. TS activity in murine bone marrow cells was strongly inhibited to 10% of the control value during 48 h. LV/5FU administration resulted in a slightly higher inhibition. No human bone marrow was available to measure TS levels. Both in mice and humans the most pronounced TS inhibition occurred in the tissue that was involved in dose-limiting toxicity. Therefore it is very likely that TS inhibition in normal tissues contributes to the toxic side-effects of 5FU treatment.     

Affinity and selectivity of PD156707, a novel nonpeptide endothelin antagonist, for human ET(A) and ET(B) receptors

6-Thioguanine (6TG) a cytostatic antimetabolite is currently used to treat patients with cancer, in particular leukemias. However, one drawback of such use is the development of 6TG resistance. Hypoxanthine-guanine phosphoribosyl transferase (Hprt) plays a crucial role in the bioactivation of 6TG. Loss of Hprt has been associated with the resistance of leukemias to 6TG chemotherapy, however, nothing has been known about the effect of Hprt status on tissue specific toxicity of 6TG in vivo. We determined the effect of Hprt status on the tissue-specific toxicity of 6TG in vivo in transgenic Hprt-deficient mice. The approximate lethal dose for Hprt-deficient mice was 23-fold higher than for the wild-type. Serum biochemical analyses of 6TG-treated wild-type mice showed elevated serum enzyme levels characteristic of liver damage whereas the levels in Hprt-deficient 6TG-treated mice were within normal physiological limits. Histopathological examination of tissues from wild-type and from Hprt-deficient mice showed contrasting spectrums of microscopic lesions. Wild-type mice had loss of hematopoietic cells from bone marrow starting at the lowest dose of 25 mg/kg 6TG whereas Hprt-deficient mice had normal bone marrow and spleen even at doses of 720 mg/kg 6TG. Wild-type mice also experienced severe loss of epithelial cells from the gastrointestinal tract starting at 50 mg/kg; however, the gastrointestinal tract of Hprt -/- mice remained unaffected. Wild-type livers revealed atrophy and necrosis at doses of 25 mg/kg 6TG although Hprt -/- livers displayed no effect until 507 mg/kg. In this study we show that Hprt-deficient mice had 6TG-resistant bone marrow and there are several other factors contributing to 6TG resistance in patients. Because variations among people exist in terms of their 6TG sensitivity, determining 6TG sensitivity of lymphocytes prior to 6TG chemotherapy and restricting treatment to 6TG-sensitive patients may improve the efficacy.     

Neuropathology of bovine herpesvirus type 5 (BHV-5) meningo-encephalitis in a rabbit seizure model

CI-994 (acetyldinaline) is an orally active anticancer drug currently in Phase 1 clinical trials. To assess its preclinical toxicity, CI-994 was administered orally as suspensions to Wistar rats (10/sex/dose) and in capsules to beagle dogs (3/sex/dose) once daily for two weeks. Doses were 1.5, 5, and 15 mg/kg for rats (9, 30, and 90 mg/m2, respectively), and 0.5, 2, and 5 mg/kg for dogs (10, 40, and 100 mg/m2, respectively). Systemic exposure was dose-proportional based on toxicokinetic analysis in dogs. Severe clinical signs and mortality occurred at the highest dose in both species beginning on Day 10. Neutropenia, lymphocytopenia, thrombocytopenia, lymphoid depletion, bone marrow hypocellularity, and testicular degeneration were observed in both species, primarily at the mid- and high-doses. Despite continued treatment, neutrophil counts in dogs returned to control levels in Week 2. Other microscopic findings in rats included splenic hematopoietic depletion at all doses and epithelial cell necrosis in various tissues at 15 mg/kg. Additional bone marrow changes in dogs involved myeloid and megakaryocyte hyperplasia at 2 mg/kg and abnormal myeloid and megakaryocyte maturation at 2 and 5 mg/kg. Except for the testicular effects in both species, all changes were reversible within a 4-week (rat) or 9-week (dog) recovery period. The results of these studies show that target organ effects of CI-994 principally involve tissues with rapidly dividing cell populations and that bone marrow suppression is the dose-limiting toxicity. CI-994 also seems to interfere with the release and/or maturation of cells in the bone marrow.     

A stable RAG1-RAG2-DNA complex that is active in V(D)J cleavage

A well-known characteristic of gamma delta T cells is that they are produced in waves during ontogeny, with cells expressing T-cell receptor V gamma 5 appearing early in fetal thymic ontogeny, followed by V gamma 6, then by other gamma delta T-cell types. In addition, evidence exists to suggest that the potential of haemopoietic precursors to generate different types of gamma delta T cells changes in ontogeny. We have used these observations as the basis for an extensive study of the potential for haemopoietic precursors isolated from fetal liver, neonatal spleen and adult bone marrow to reconstitute severe combined immunodeficient (SCID) mice. Mice that were reconstituted as newborns with fetal liver cells most closely resembled normal C.B-17 mice with respect to both lymphocyte numbers and subsets, while mice reconstituted with adult bone marrow had fewer cells than normal mice. This deficit spanned both T and B cells in all organs examined. Among the gamma delta T-cell subsets examined, the ability to reconstitute V gamma 4+ cells was particularly dependent on the ontogenic age of the reconstituting presursors, with fetal liver cells having the greatest capacity to generate V gamma 4+ cells, and adult bone marrow cells the least. The vast majority of the T cells produced in the reconstituted mice were of donor origin, and the level of reconstitution was found to be dependent upon some factor other than the presursor frequency.     

Activity of liposomal nystatin against disseminated Aspergillus fumigatus infection in neutropenic mice

The purpose of this study was to examine the activity of liposomal nystatin against a disseminated Aspergillus fumigatus infection in neutropenic mice. Mice were made neutropenic with 5-fluorouracil and were administered the antifungal drug intravenously for 5 consecutive days beginning 24 h following infection. Liposomal nystatin, at doses as low as 2 mg/kg of body weight/day, protected neutropenic mice against Aspergillus-induced death in a statistically significant manner at the 50-day time point compared to either the no-treatment, the saline, or the empty-liposome group. This protection was approximately the same as that for free nystatin, a positive control. Histopathological results showed that liposomal nystatin cleared the lungs, spleen, pancreas, kidney, and liver of Aspergillus and that there was no organ damage at the day 5 time point, which was after only three doses of liposomal nystatin. Based on these results in mice, it is probable that liposomal nystatin will be effective against Aspergillus infection in humans.     

The molecular mechanisms of 5-fluoro-2'-deoxyuridine induced cell death

The molecular mechanism of cell death induced by 5-Fluoro-2'-deoxyuridine (FUdR) was investigated. FUdR caused cell death to induce dNTP pool imbalance and following DNA double strand breaks in mouse mammary tumor FM3A cells. We isolated a new endonuclease from FUdR-treated cells, named endonuclease S, that played an important role in FUdR-induced cell death. Cells treated with FUdR showed intracellular acidification before cell death formation. We observed that the endonuclease S in acidic cells may lead the DNA fragmentation. On the other hand, we observed that protease inhibitors (such as TLCK, TPCK, PMSF, p-APMSF, Pefabloc SC and Z-Asp-CH2-DCB) blocked intracellular acidification, DNA fragmentation and FUdR-induced cell death. But the inhibitors did not affect dNTP pool imbalance in the cells. These results suggest that proteases act at the point of downstream of dNTP pool imbalance and upstream of the intracellular acidification.     

Keratinocyte growth factor administered before conditioning ameliorates graft-versus-host disease after allogeneic bone marrow transplantation in mice

Keratinocyte growth factor (KGF) is important in tissue repair and wound healing and its administration can abrogate chemical- and radiation-induced tissue damage in rodents. We investigated KGF as a therapeutic agent for the prevention of graft-versus-host disease (GVHD)-induced tissue damage, morbidity, and mortality in an established murine allogeneic bone marrow transplantation (BMT) model. B10.BR (H2(k)) recipient mice were lethally irradiated and transplanted with C57BL/6 (H2(b)) bone marrow (BM) with spleen cells (BMS) as a source of GVHD-causing T cells. KGF-treated mice (5 mg/kg/d subcutaneously days -6, -5, and -4 pre-BMT) receiving BMS exhibited better survival than those not receiving KGF (P =.0027). Cyclophosphamide (Cy), a common component of total body irradiation (TBI)-containing regimens, was administered to other cohorts of mice at a dose of 120 mg/kg/d intraperitoneally on days -3 and -2 before BMT. KGF-treated mice again exhibited a better survival rate than those not receiving KGF (P =.00086). However, KGF-treated recipients receiving TBI or Cy/TBI BMS were not GVHD-free, as shown by lower body weights compared with BM groups. GVHD target tissues were assessed histologically during a 38-day post-BMT observation period. KGF ameliorated GVHD-induced tissue damage in the liver, skin, and lung (completely in some recipients) and moderately so in the spleen, colon, and ileum, even with Cy conditioning. These studies demonstrate that KGF administration, completed before conditioning, has potential as an anti-GVHD therapeutic agent.     

Response of mosquitoes (Diptera: Culicidae) to carbon dioxide and octenol in Egypt

The radioprotective effect of the leaf extract of Ocimum sanctum (OE) in combination with WR-2721 (WR) was investigated on mouse bone marrow. Adult Swiss mice were injected intraperitoneally (i.p.) with OE (10 mg/kg on 5 consecutive days), or 100-400 mg/kg WR (single dose) or combination of the two or double-distilled water (DDW) and whole-body exposed to 4.5 Gy gamma-irradiation (RT). Metaphase plates were prepared from femur bone marrow on days 1, 2, 7 and 14 post-treatment and chromosomal aberrations were scored. The maximum number of aberrant cells was observed at 24 h after irradiation in all the groups. However, pretreatment with OE or WR individually resulted in a significant decrease in aberrant cells as well as different types of aberrations. The combination of the two further enhanced this effect; resulting in a 2-fold increase in the protection factor (PF = 6.68) compared to 400 mg/kg WR alone. The percent aberrant cells decreased linear-quadratically with WR dose when given individually, while in the OE + WR pretreatment animals the values showed a linear dose response. Combination of OE with WR doses above 200 mg/kg completely eliminated rings, polyploidy and pulverization of chromosomes. Percent aberrant cells decreased with time in all groups, though the values remained higher than normal even on day 14 in the RT alone as well as those treated with single agent + RT. WR doses above 200 mg/kg before RT resulted in significantly higher frequency of aberrant cells compared to RT and OE + RT groups on day 14, suggesting delayed WR toxicity; but combination of OE with WR brought down these values to normal level, indicating that OE combination, in addition to enhancing WR protection, may also act as a detoxifier. The protective effect of OE and WR is also reflected in the enhancement of bone marrow CFU survival. Both OE and WR possessed significant free radical scavenging activity in vitro. The combination of the two further enhanced this effect, suggesting that the enhanced free radical scavenging activity by combining the two protectors results in the higher bone marrow cell protection. The significant elevation in chromosome protection obtained by combining OE with WR, with reduction in the latter's toxicity at higher doses, suggests that the combination may have promise for radioprotection in humans.     

Comparison of the toxicity profiles of ISIS 1082 and ISIS 2105, phosphorothioate oligonucleotides, following subacute intradermal administration in Sprague-Dawley rats

The systemic toxicity of two phosphorothioate oligonucleotides specific for herpes simplex viruses (ISIS 1082) and human papiloma virus (ISIS 2105) were evaluated following repeated intradermal injections of vehicle control, 0.33, 2.17, or 21.7 mg/kg daily to Sprague-Dawley rats (10/sex/group) for 14 days. Animals were sacrificed 1 day after the last dose, except for a portion of the ISIS 1082-treated animals (5/sex/group) which were maintained for an additional 14-day recovery period. The profile of alterations noted for both compounds was very similar. Other than local signs of irritation at the site of injection, there were no clinical signs of toxicity or treatment-related mortality, but there was a slight decrease in body weight gain for the 21.7 mg/kg dose groups. Alterations in hematology parameters included dose-dependent thrombocytopenia and anemia. Alterations in serum chemistry parameters were suggestive of mild alterations in hepatic metabolism, with increases in liver transaminases and bilirubin, along with decreases in albumin and cholesterol. Both spleen and liver weights were significantly elevated in a dose-dependent fashion. Histopathological alterations noted in liver, kidney, lung, injection site skin, and spleen were characterized as perivascular and interstitial infiltrates of macrophages and monocytes. Additional microscopic alterations in the spleen included mild lymphoid hyperplasia (seen in lymph nodes as well), and extramedullary hematopoiesis. Treatment-related cytopenias were likely related to mild, focal hypocellularity in the bone marrow. Alterations in ISIS 1082-treated animals were only partially reversed following the 14-day treatment-free period. In conclusion, repeated intradermal administration of ISIS 1082 and ISIS 2105 produced a similar spectrum of toxicities, with liver, kidney, spleen, and bone marrow being identified as target tissues.     

Adenosine affects the f-met-leu-phe induced changes in cytosolic Ca2+ and in membrane potential of human granulocytes

The effect of administering the thiol modulating agent buthionine sulfoximine (BSO) in conjunction with alkylating chemotherapy was investigated in vivo in the mouse KHT sarcomas and bone marrow stem cells. Tumour response to treatment was assessed by an in vivo to in vitro excision assay and bone marrow survival was determined in vitro by CFU-GM. Glutathione (GSH) depletion and recovery kinetics were determined at various times after treatment using high performance liquid chromatography (HPLC) techniques. Following a single 2.5 mmol kg-1 dose of BSO, tumour GSH reached a nadir of approximately 40% of control 12-16 h after treatment. Bone marrow GSH was depleted to approximately 45% of control 4-8 h after treatment but recovered to normal by 16 h. When a range of doses of CCNU, mitomycin C, cyclophosphamide or melphalan (MEL) were given 16 h after mice were exposed to a 2.5 mmol kg-1 dose of BSO, only the antitumour efficacy of MEL was effectively enhanced (by a factor of approximately 1.4). This BSO-MEL combination appeared to be selective for the tumour as the bone marrow toxicity was not increased beyond that seen for MEL alone. Since increasing the administered dose of BSO neither increased the extent of thiol depletion in the tumour nor enhanced the antitumour efficacy of MEL, three other protocols for delivering the thiol depletor were explored. BSO was given either as multiple 2.5 mmol kg-1 doses administered at 6 or 16 h intervals or continuously at a concentration of 30 mM supplied in the animals' drinking water. Both multi-dose BSO pretreatments were found to increase both the antitumour efficacy and normal tissue toxicity of MEL such that no advantage compared to the single dose combination was achieved. In contrast, maintaining the thiol depletor in the drinking water led to an approximately 1.7-fold increase in the antitumour efficacy of MEL without any corresponding increase in bone marrow stem cell toxicity. For the various pretreatment strategies it was possible, in all cases, to account for the presence or absence of a net therapeutic benefit on the basis of the tumour and bone marrow GSH depletion and recovery kinetics.     

Characterization of the growth factor activity of amniotic fluid on cells from hematopoietic and lymphoid organs of different life stages

We investigated the proliferation-promoting effects of murine amniotic fluid (MAF) on in vitro cultured cells originally obtained from murine hematopoietic and lymphoid organs at different life stages. MAF promoted proliferation of the fetal liver cells (FLC), newborn spleen cells and adult bone marrow cells. The proliferation-promoting activity of MAF was extended to liver cells and spleen cells from mice younger than 2 weeks old. MAF did not, however, promote the proliferation of newborn or adult thymocytes, or of spleen cells, liver cells or peritoneal cells, liver cells or peritoneal cells from 2-week-old or older mice. Rather, it partially inhibited the proliferation of spleen cells, thymocytes and peritoneal cells from 1-year-old mice. These results suggest that MAF contains growth factors for hematopoietic stem cells but not for either mature or immature T lymphocytes. Supporting this view, the MAF activity was partially neutralized by a polyclonal anti-mouse stem cell factor (SCF) antibody. Moreover, the immunoblotting of MAF against anti-mouse SCF antibody revealed a band at 30-32 kDa corresponding to the previously reported SCF. Interestingly, MAF was able to maintain FLC and adult bone marrow cells alive in culture for a relatively long time (2 weeks). The MAF activity was further shown to be partially and cell type-dependently antagonized by TNF-alpha and TGF-beta. These results provided evidence that MAF contains potentially multiple growth factors preferentially affecting the early stage of hematopoiesis, one of which is SCF.     

Series: current issues in mutagenesis and carcinogenesis, No. 65. The genotoxicity and carcinogenicity of paracetamol: a regulatory (re)view

The publication of several studies reporting genotoxic effects of paracetamol, one of the world's most popular over-the-counter drugs, has raised the question of regulatory action. Paracetamol does not cause gene mutations, either in bacteria or in mammalian cells. There are, however, published data giving clear evidence that paracetamol causes chromosomal damage in vitro in mammalian cells at high concentrations and indicating that similar effects occur in vivo at high dosages. Available data point to three possible mechanisms of paracetamol-induced genotoxicity: (1) inhibition of ribonucleotide reductase; (2) increase in cytosolic and intranuclear Ca2+ levels; (3) DNA damage caused by NAPQI after glutathione depletion. All mechanisms involve dose thresholds. Studies of the relationship between genotoxicity and toxic effects in the rat (induction of micronuclei in rat bone marrow including dose-response relationship, biotransformation of paracetamol at different dosages, concomitant toxicity and biochemical markers) have recently been completed. These studies, which employed doses ranging from the dose resulting in human therapeutic peak plasma levels to highly toxic doses, give convincing evidence that genotoxic effects of paracetamol appear only at dosages inducing pronounced liver and bone marrow toxicity and that the threshold level for genotoxicity is not reached at therapeutic dosage. Reliable studies on the ability of paracetamol to affect germ cell DNA are not available. However, based on the amount of drug likely to reach germ cells and the evidence of thresholds, paracetamol is not expected to cause heritable damage in man. Various old and poorly designed long-term studies of paracetamol in the mouse and rat have given equivocal results. A few of these studies showed increased incidence of liver and bladder tumours at hepatotoxic doses. National Toxicology Program (U.S.A.) feeding studies have shown that paracetamol is non-carcinogenic when given at non-hepatotoxic doses up to 300 mg/kg/d to the rat and up to 1000 mg/kg/d to the mouse. Taking into account the knowledge of the hepatotoxicity and metabolism of paracetamol and the existence of thresholds for its genotoxicity, the animal studies do not indicate a carcinogenic potential at non-hepatotoxic dose levels. Based on this updated assessment of the genotoxicity and carcinogenicity of paracetamol, it is concluded that there is no need for regulatory action.     

Steroid-responsive interstitial lung disease in patients receiving 2'-deoxy-5-fluorouridine-infusion chemotherapy. A report of three cases

BACKGROUND: Continuous infusion of 2'-deoxy-5-fluorouridine (FUdR) has shown promise in its activity against metastatic renal cell carcinoma. Its side-effect profile is dominated by gastrointestinal toxicity; there are no known adverse pulmonary reactions. To the authors' knowledge, this is the first report on the development of lung toxicity in three patients receiving FUdR-infusion chemotherapy for metastatic renal cell carcinoma. METHODS: The hospital charts of three patients presenting with pulmonary symptoms during FUdR chemotherapy were reviewed. A literature search was performed regarding FUdR-related pulmonary toxicity. RESULTS: Nonproductive cough, dyspnea, and fever appeared within the 10th chemotherapy cycle. Chest radiographs showed interstitial disease in each case, accompanied by a restrictive pattern by pulmonary-function testing. Lung biopsies were performed on two patients showing a pattern of interstitial inflammation. Discontinuing FUdR and instituting steroidal therapy invariably improved symptoms, as was evident by chest radiographs and pulmonary function tests. In one patient, resuming FUdR treatment resulted in a recurrence of the respiratory symptoms, which was controlled with an increased steroidal dose. All three patients required low dose steroids to maintain their baseline respiratory functions. CONCLUSIONS: 2'-deoxy-5-fluorouridine-related lung toxicity is an uncommon event and occurs late in the treatment course. It is rapidly symptomatic and responds readily to steroidal therapy.     

An in vivo/in vitro method for assessing micronucleus and chromosome aberration induction in rat bone marrow and spleen. 2. Studies with chlorambucil and mitomycin C

An in vivo/in vitro system using rat bone marrow cells and spleen cells to assess micronucleus (MN) and structural chromosome aberrations (SCA) simultaneously (Moore et al., 1995) was further developed. In two separate experiments, two rats/dose/experiment were treated i.p. with 0, 5, 10 and 15 mg chlorambucil (CA)/kg or with mitomycin C (MMC) at 0, 1, 2, 4 mg/kg (experiment 1) or 0, 4, 6, and 8 mg/kg (experiment 2) and killed 6 h later. Cultures were then established in the presence of growth stimulants (interleukin-3 and granulocyte-macrophage colony stimulating factor for bone marrow; lipopolysaccharide and concanavalin A for spleen) and cytochalasin B, a cytokinesis inhibitor. Bone marrow cells were harvested 24 h after establishment of cultures, while spleen cells were harvested at 48 h. In addition, spleen cells were concurrently assayed for chromosome aberrations. With the MN endpoint, spleen cells appeared more sensitive than bone marrow cells to the effects of CA due both to a lower background and an increased response. For MMC, bone marrow cells exhibited both a higher background of MN and a greater numerical response than did spleen cells. However, on the basis of a fold-increase over control values, spleen cells were more sensitive than bone marrow cells. In general, the MN endpoint appeared more sensitive than the SCA in spleen cells after treatment with CA or MMC. Thus, the approach described here shows greater potential in detecting genotoxicity.     

Evaluation of the rat micronucleus test with bone marrow and peripheral blood: summary of the 9th collaborative study by CSGMT/JEMS. MMS. Collaborative Study Group for the Micronucleus Test. Environmental Mutagen Society of Japan. Mammalian Mutagenicity Study Group

The mouse has traditionally been used for the micronucleus test, with bone marrow the usual target organ. The aim of the 9th collaborative study by CSGMT was to evaluate the suitability of the rat for the micronucleus test, with bone marrow and peripheral blood as the target organ. Since the rat spleen eliminates circulating micronucleated erythrocytes, a rat peripheral blood micronucleus assay might not be feasible. Thirty-four Japanese laboratories and six overseas laboratories participated in this collaboration, and 40 chemicals were studied. As a rule, rat bone marrow and peripheral blood were analyzed using acridine orange staining. Among 36 mouse micronucleus-positive rat carcinogens, 34 of which had been evaluated by CSGMT, we observed 33 positive and three negative results with rat bone marrow and 30 positive, three equivocal, and three negative responses with rat peripheral blood. Of the two mouse micronucleus-negative rat carcinogens, acrylonitrile was positive in rat bone marrow and 4,4'-methylene bis(2-chloroaniline) was negative in both rat bone marrow and peripheral blood. Two chemicals reported to be mouse micronucleus-negative and rat-positive, azobenzene and Solvent Yellow 14, and one chemical reported to be mouse-positive and rat-negative, 1,2-dimethylhydrazine, gave positive responses in rat bone marrow and peripheral blood. The concordance between bone marrow and peripheral blood with rats was 92%. The concordance between rat and mouse erythrocytes was 88%. We concluded that the rat micronucleus assay, using either bone marrow or peripheral blood, can be used as an alternative to the mouse micronucleus assay.     

Thermoperiodic responses in insects and mites simulated with the double circadian oscillator clock

PURPOSE: To establish the pharmacodynamic relationships between drug biodistribution and drug toxicity/efficacy, a comprehensive preclinical evaluation of sphingomyelin/cholesterol (SM/chol) liposomal vincristine and unencapsulated vincristine in mice was undertaken. METHODS: Pharmaceutically acceptable formulations of unencapsulated vincristine and liposomal vincristine at drug/lipid ratios of 0.05 or 0.10 (wt/wt) were evaluated for toxicity, antitumor activity and pharmacokinetics following intravenous administration. RESULTS: Mice given liposomal vincristine at 2 mg/kg vincristine had concentrations of vincristine in blood and plasma at least two orders of magnitude greater then those achieved after an identical dose of unencapsulated drug. One day after administration of the liposomal vincristine, there were at least tenfold greater drug quantities, relative to unencapsulated vincristine, in the axillary lymph nodes, heart, inguinal lymph nodes, kidney, liver, skin, small intestines and spleen. Increased plasma and tissue exposure to vincristine as a result of encapsulation in SM/chol liposomes was not associated with increased drug toxicities. Treatment of the murine P388 ascitic tumor with a single intravenous dose of unencapsulated drug at 2, 3 and 4 mg/kg, initiated 1 day after tumor cell inoculation, resulted in a 33 to 38% increase in lifespan. In contrast, long-term survival rates of 50% or more were achieved in all groups treated with the SM/chol liposomal vincristine formulations at doses of 2, 3 and 4 mg/kg. At the 4 mg/kg dose, eight of ten and nine of ten animals survived past day 60 when treated with SM/chol liposomal vincristine prepared at the 0.05 and 0.1 drug/lipid ratios, respectively. CONCLUSIONS: Overall, increased and prolonged plasma concentrations of vincristine achieved by liposomal encapsulation were correlated with dramatically increased antitumor activity in comparison with the unencapsulated drug, but no correlations could be established between pharmacokinetic parameters and toxicity.