Median LC50 Research Articles

Analysis of eight oil spill dispersants using rapid, in vitro tests for endocrine and other biological activity.

The Deepwater Horizon oil spill has led to the use of >1 M gallons of oil spill dispersants, which are mixtures of surfactants and solvents. Because of this large scale use there is a critical need to understand the potential for toxicity of the currently used dispersant and potential alternatives, especially given the limited toxicity testing information that is available. In particular, some dispersants contain nonylphenol ethoxylates (NPEs), which can degrade to nonylphenol (NP), a known endocrine disruptor. Given the urgent need to generate toxicity data, we carried out a series of in vitro high-throughput assays on eight commercial dispersants. These assays focused on the estrogen and androgen receptors (ER and AR), but also included a larger battery of assays probing other biological pathways. Cytotoxicity in mammalian cells was also quantified. No activity was seen in any AR assay. Two dispersants showed a weak ER signal in one assay (EC50 of 16 ppm for Nokomis 3-F4 and 25 ppm for ZI-400). NPs and NPEs also had a weak signal in this same ER assay. Note that Corexit 9500, the currently used product, does not contain NPEs and did not show any ER activity. Cytotoxicity values for six of the dispersants were statistically indistinguishable, with median LC50 values approximately 100 ppm. Two dispersants, JD 2000 and SAF-RON GOLD, were significantly less cytotoxic than the others with LC50 values approaching or exceeding 1000 ppm.

In-Vitro Efficacy of the Deoxyguanoside Analogs Forodesine (BCX-1777) and ARA-G in Pediatric Acute Lymphoblastic Leukemia.

Abstract 2038Poster Board II-15Purine nucleoside phosphorylase (PNP) deficiency in humans is associated with elevated deoxyguanosine (dGuo) plasma levels. DGuo is converted into dGTP inducing apoptosis in T-cells and this provides the rationale for the development of deoxyguanosine analogues as a potential treatment option for T-cell malignancies. Forodesine (BCX-1777; BioCryst-Mundipharma) is an efficient blocker of PNP activity, thereby boosting the conversion of dGuo into dGTP and raising intracellular dGTP levels. AraG (9-b-D-arabinofuranosyl-guanine) is a compound that is resistant to PNP-mediated degradation that is efficiently converted into AraGTP. AraGTP becomes incorporated in the DNA, blocking DNA synthesis and promoting apoptosis. In a phase II clinical trial, the AraG prodrug Nelarabine enforced a complete remission rate of 55% for pediatric T-ALL patients at 1st relapse. (Berg, JCO 2005). Clinical data of Forodesine treatment in pediatric ALL patients are not yet available.As tested on primary pediatric acute lymphoblastic leukemia (ALL) patient samples (4 T-ALL, 2 BCP-ALL), 1μM of Forodesine is sufficient to completely block PNP and abolish rapid dGuo degradation resulting in a median 7.9 (range 0.5-378) fold raise of intracellular dGTP levels. Accumulation of dGTP is comparable for T-ALL (n=31) and BCP-ALL (n=11) patient samples. This reflects equal intrinsic ability of salvage nucleotide synthesis for both T-ALL and BCP-ALL cells. Cytotoxic effect of Forodesine was tested on primary leukemia cells from newly diagnosed pediatric ALL patients in-vitro by incubating cells with Forodesine (1μM) in the presence of increasing concentrations of dGuo (0.001-50μM). In accordance with selective T-cell toxicity, T-ALL cells were more sensitive to Forodesine/dGuo treatment (median T-ALL LC50 value: 1.1μM dGuo/1μM Forodesine, n=27, p=0.001) compared to BCP-ALL cells, which had a median LC50 value of 8.8μM dGuo/1μM Forodesine (n=30). All patients that responded demonstrated dGTP accumulation (1.5-222.1 fold), although the raise of dGTP levels did not correlate with Forodesine/dGuo toxicity (r2= 0.10, p=0.22).Studying in-vitro responsiveness to AraG, T-ALL cells were more sensitive compared to BCP-ALL cells (p=0.0002) with a median AraG LC50 value of 20.5μM for T-ALL samples (n=24) versus 48.3μM for BCP-ALL samples (n=20). Remarkably, TELAML1 positive BCP-ALL cases were insensitive to AraG treatment (median LC50 value >50μM, n=9). No correlation was identified between in-vitro Forodesine/dGuo and AraG cytotoxicities (r2=0.05, p=0.29). Most patient samples that displayed AraG resistance still responded to Forodesine/dGuo treatment. This may be explained by the fact that the uptake of both drugs may be facilitated by different transporters. Using RQ-PCR we could demonstrate that AraG toxicity, in contrast to Forodesine, was significantly associated with ENT1 (equilibrative nucleoside transporter 1) expression levels (p=0.008), which was previously identified as strong predictor for AraC cytotoxicity in pediatric ALL (Stam RW. et al., Blood 2003). AraG cytotoxicity strongly correlated with AraC cytotoxicity (r2=0.71, p<0.0001). We found no significant correlation between Forodesine sensitivity and the expression levels of other nucleoside transporters (CNT1, CNT2, CNT3, ENT2), kinases (dCK, dGK), nucleotidases (NT5C1A, NT5C2, PNI) or other enzymes that are involved in dGuo metabolism (PNP, RRM1, RRM2).In conclusion, T-ALL cells are more sensitive to Forodesine/dGuo treatment in-vitro than BCP-ALL cells that have nearly 8 fold higher dGuo LC50 values. Resistance to AraG treatment does not preclude responsiveness to Forodesine treatment and vice versa, indicating that Forodesine and AraG rely on different cellular mechanisms for cytotoxicity, possibly involving differences in dependence on the nucleoside transporter ENT1. Disclosures:No relevant conflicts of interest to declare.

In-Vitro Efficacy of the Deoxyguanoside Analogs Forodesine (BCX- 1777) and ARA-G in Pediatric Acute Lymphoblastic Leukemia Cells.

Purine nucleotide phosphorylase (PNP) deficiency in humans is associated with elevated dGuo plasma levels. This results in the intra-cellular conversion of dGuo into dGTP, following 3 consecutive kinase steps and depletion of T-cells resulting in immune deficiency. This T-cell toxicity provided the rationale for the development of deoxyguanosine analogues as potential therapeutic compounds for T-cell malignancies. Forodesine (BCX-1777; BioCryst-Mundipharma) is an efficient blocker of PNP activity. Forodesine facilitates the conversion of dGuo into dGTP raising the intracellular dGTP pool. AraG (9-b-D-arabinofuranosyl-guanine) is a compound that is resistant to PNP-mediated degradation resulting in phosphorylation of AraG into AraGTP. AraGTP becomes incorporated in the DNA and blocks DNA synthesis resulting in apoptosis. In a phase II clinical trial, the AraG prodrug Nelarabine enforced a complete remission rate of 55% for pediatric T-ALL patients at 1st relapse. (Berg, JCO 2005). Clinical data of forodesine treatment in pediatric ALL patients are not yet available.The cytotoxic effect of Forodesine was investigated on primary leukemia cells from newly diagnosed pediatric acute lymphoblastic leukemia (ALL) patients in-vitro. Cells were incubated with a fixed concentration of Forodesine (1μM) in the presence of increasing concentrations of dGuo (0.001–50μM). The dGTP levels under conditions where PNPactivity was completely blocked was monitored.Incubation of primary leukemic cells obtained from 6 pediatric ALL patients (4 T-ALL, 2 B-ALL) with 10μM dGuo results in rapid dGuo degradation (t½<4hrs) by the PNP enzyme that is completely abolished by the addition of 1μM of Forodesine. Cells consequently accumulate dGTP levels upon Forodesine treatment to a median 7.9 (range 0.5–378 fold) that is comparable between T-ALL (n=31) and B-ALL (n=11) patient samples. This reflects equal intrinsic ability of de-novo nucleotide synthesis for both T-ALL and B-ALL cells. In accordance with T-cell selective toxicity, T-ALL cells were more sensitive to Forodesine/ dGuo treatment (median T-ALL LC50 value: 1.1μM dGuo/1μM Forodesine, n=27, p=0.001) compared to B-ALL cells, which had a median LC50 value of 8.8μM dGuo/1μM Forodesine (n=30). All patients that responded demonstrated dGTP accumulation (1.5– 222.1 fold), but the magnitude of dGTP accumulation did not relate to Forodesine/dGuo toxicity.Studying in-vitro responsiveness to AraG, T-ALL cells were more sensitive compared to B-ALL cells (p=0.0002) with a median AraG LC50 value of 20.5μM for T-ALL samples (n=24) versus 48.3μM for B-ALL samples (n=20). However, TELAML1 negative B-ALL cases were sensitive to AraG where as TELAML1 positive B-ALL cases were remarkable insensitive to AraG treatment (median LC50 value >50μM, n=9). No correlation was identified between in-vitro Forodesine/dGuo and AraG cytotoxicities. Most patient samples that displayed AraG resistance still responded to Forodesine/dGuo treatment.In contrast, AraG cytotoxicity strongly correlated with AraC cytotoxicity (r2=0.71, p<0.0001). In conclusion, T-ALL cells are sensitive to Forodesine/dGuo treatment in-vitro in contrast to B-ALL cells that have nearly 8 fold higher LC50 values. In-vitro Forodesine mediated cytotoxicity seems more potent in pediatric ALL than AraG treatment. Resistance to AraG treatment does not preclude responsiveness to Forodesine treatment and vice versa, indicating that Forodesine and AraG rely on different cellular mechanisms for cytotoxicity.

The Effect Of Textile Mill Effluent On Two Brachish Fauna Species (Oreochromis Niloticus And Palaemonetes Africanus) In Ibeshe River Lagos State

Acute toxicity of textile mill effluent to two brackish fauna species, Oreochromis niloticus (finfish) and Paleamonetes africanus. (Shrimp) fingerlings were assessed in a static renewable bioassay for 96h to determine median lethal concentrations (LC50). Seven different graded concentrations were prepared as 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 and 2, 4, 6, 8, 10, 12mg L-1 for Oreochromis niloticus and Paleamonetes africanus fingerlings, respectively. However, a control experiment (0mgL-1) was set up accordingly. The 96h (LC50) value computed on logarithm were 1.21 and 5.11mgL-1 for Oreochromis niloticus and Paleamonetes africanus fingerlings, respectively. The median LC50 were 24, 48, 72 and 96h for 4.83, 2.01, 1.38 and 1.20 mgL-1 on Oreochromis niloticus fingerlings, exposed to different concentration textile mill effluent. However, median LC50 were 24, 48, 72 and 96h for 9.02, 7.82, 6.76, and 5.09 mgL-1 on Paleamonetes africanus fingerlings, exposed to different concentrations of textile mill effluent. The fingerlings of tilapia and shrimps exposed to various concentration of effluent showed uncoordinated behaviour, increase in their movements, ventilation, spiral swimming, sideway swimming, effort to swallow air from the surface for the tilapia fingerlings while shrimp fingerlings showed backward swimming and static swimming for few minutes. Mortality was recorded early in Oreochromis niloticus fingerlings at 0.5mgL-1 during a 24h exposure to the textile mill effluent while for the Paleamonetes africanus, mortality was recorded at 6mg L-1 for 24h exposure to the textile mill effluent physico-chemical assessment of the textile mill effluent were given as: Total Suspended Solids TSS (400-mgL-1) Biological Oxygen Demand BOD (362.6-397.1 mgL-1, Detergent (40.2-49.9 mgL-1) Alkalinity (575-1225 mgL-1), Oil and grease 28.3-31.2 mgL-1), Dissolved Oxygen DO (3.30-3.90 mgL-1). These were higher than the FEPA limit for effluent discharge. However, physico chemical analysis revealed low, species diversity of aquatic fauna in Ibeshe River. This suggests that effluent discharge into the river is hig7hly toxic to brackish water fauna species. Keywords: Toxicity, effluent, Oreochromis niloticus, Paleamonetes africanus, physico-chemical parameters. JOAFSS Vol. 4 (1) 2006: pp. 194-203

Ex Vivo Activity of Bortezomib and Dexamethasone Combinations Against Childhood Acute Leukemia Cells.

Response to glucocorticoid (GC) treatment is of prognostic value for children with acute lymphoblastic leukemia (ALL). In this context, sensitization of leukemic cells for GC may further improve the prognosis. In recent studies (Oerlemans et al, 2007) we showed that chronic inhibition of the nuclear transcription factor NFkB sensitized leukemia cells for GC treatment, including GC-resistant acute myeloid leukemia (AML) cells. The proteasome inhibitor bortezomib (BTZ) exerts part of its mechanism of action via inhibition of NFkB activation. The aim of our study was to determine the cytotoxic effects of BTZ, the GC dexamethasone (DEX), and their combination, against childhood leukemia cells and normal bone marrow (BM) cells as controls. Drug (combination) responses in samples of childhood leukemia and normal BM cells were evaluated after a 4 day drug exposure using the MTT-cytotoxicity assay. The drug concentration that was lethal to 50% of cells compared to control cells without drugs was defined as LC50. Synergism between the 2 drugs was established when the percentage of leukemic cell survival (LCS) of the combination was smaller than the % LCS BTZ x % LCS DEX/100. Inclusion criteria for the MTT analysis were a percentage of blasts present in the leukemic samples > 80%, and a MTT-OD read-out of control cells at day 4 minus the blank control > 0.060. Overall analyses included 5 normal BM samples, 18 ALL and 9 AML pediatric patient samples. All normal BM cells were insensitive to DEX (LC50 values > 15.3 uM). AML cells were, as expected, significantly (p<0.01) more resistant to DEX (median LC50 >15.3 uM) as compared to ALL cells (LC50 = 0.41 uM). Within the group of AML samples 8 out of 9 (89%) had an LC50 > 15.3 uM, while only 6 of 18 (34%) of the ALL samples had such a high LC50 value. For BTZ cytotoxicity similar differences were found. With a median LC50 of 14.6 nM (range: 10–236 nM), normal BM samples were significantly (p<0.01) less sensitive for BTZ than ALL cells (median LC50 5.7 nM, range: 4–10 nM). BTZ-sensitivity of AML cells (median LC50: 14.6 nM, range: 10–26 nM) was not significantly different from normal BM cells. Based on the dose-response analysis of BTZ, 4 different concentrations of BTZ were used (ranging from 0.3 to 34 nM) in combination with 6 concentrations of DEX (ranging from 0.0046 to 15.3 uM). The drug combination predominantly revealed an additive effect, although a low level of synergism was observed at the highest concentration of BTZ and those patient samples that were resistant to DEX. This particularly referred to 6 of 9 (67%) AML samples but was also noted in 2 of 5 (40%) normal BM samples. 3 of 18 (17%) ALL samples showed synergism for the drug combination. In conclusion, BTZ displayed potent cytotoxic effects against pediatric ALL cells. This antileukemic effect may be further enhanced in combination with DEX, based on frequent additive and synergistic drug interactions we observed. The combination of BTZ and DEX is therefore a candidate drug combination for further clinical evaluation in the treatment of especially childhood ALL.

2-Phenylacetylenesulfonamide Upregulates Noxa and Induces p53-Independent Apoptosis of CLL Cells.

The classical pathway of p53-mediated apoptosis involves transcriptional upregulation of pro-apoptotic proteins including Puma and Noxa. However, recent studies have identified a novel non-transcriptional mechanism mediated by direct binding of p53 to Bcl-2 or Bcl-XL at the mitochondrial surface and consequent neutralization of their anti-apoptotic function (Chipuk and Green, Cell Cycle , 2004; 3: 429–431). Induction of apoptosis of chronic lymphocytic leukaemia (CLL) cells by cytotoxic agents is strongly dependent on a functional p53 system and genetic changes which compromise p53 function result in drug resistance. We are therefore attempting to identify novel therapeutic agents which induce selective apoptosis of CLL cells by mechanisms independent of p53. Here we present data suggesting that 2-phenylacetylenesulfonamide (PAS; trivial name pifithrin μ) is a potential therapeutic agent for CLL. PAS was toxic to CLL cells, with a median LC50 of 10.72 μM (SD 3.8 μM n=20) as measured by a dye reduction cytotoxicity assay. Killing of CLL cells was by apoptosis, documented by the ability of 10–20 μM PAS to induce cytochrome c release from mitochondria (Fn2) to the cytosol (Fn1; Figure 1, upper), conformational change of Bax to a pro-apoptotic conformation, translocation of Bax from the cytosol to mitochondria (Figure 1, lower) and cleavage of the caspase 3 substrate poly(ADP ribose) polymerase (PARP). PAS toxicity was p53-independent, since p53-deleted CLL isolates were susceptible to killing. Furthermore, PAS was toxic towards leukemic cell lines with either inducible (Daudi), non-functional (Raji) or deleted (K562) p53. PAS treatment of CLL cells resulted in upregulation of Noxa and the subsequent displacement by Noxa of the pro-apoptotic Bim long (L) and extra-long (EL) isoforms from Bcl-2, as determined by western blot analysis of Bcl-2 immunoprecipitates (Figure 2). These observations contrast with those of Strom et al (Nat Chem Biol , 2006; 2: 474–479), who reported that PAS caused the displacement of p53 from mitochondria to the cytosol and thereby protected the cells from p53-dependent apoptosis. These differences may result from ectopic viral expression of p53 in the latter study whereas this protein was under normal cellular regulation in CLL cells. Normal T lymphocytes were relatively resistant to PAS, with IC50 values four times greater than those observed for CLL cells. In conclusion, our data suggest that PAS represents a novel class of agent which is toxic towards CLL cells via a mechanism involving p53-independent upregulation of Noxa and the subsequent unleashing of pro-apoptotic Bim from anti-apoptotic Bcl-2. This p53-independent toxicity may be of particular value in the treatment of drug-resistant CLL patients with compromised p53 pathways. [Display omitted] [Display omitted]

Addition of PTK787/ZK 222584 Can Lower the Dosage of Amsacrine To Achieve Equal Amounts of AML Cell Death.

Acute myeloid leukemia (AML) is a disease with a poor prognosis. At this moment conventional chemotherapy is the gold standard for the treatment of AML. It is demonstrated that AML cells express VEGFA and VEGFC as well as KDR (VEGFR2), the main receptor for downstream effects, resulting in an autocrine pathway for cell survival. The present study investigates the role of PTK787/ZK 222584, a potent inhibitor of VEGF receptor tyrosine kinases, upon leukemic cell death, and the possibility of an additional effect upon cell death achieved by a conventional chemotherapeutic drug Amsacrine. In 3 AML-cell lines and 33 pediatric AML patient samples we performed total cell kill assays to determine the percentage of cell death achieved by PTK787/ZK 222584 (5–100 μM) and/or Amsacrine (0.001–2 μg/ml). Both drugs induced AML cell death. The LC50 values (drug concentration needed to kill 50% of the leukemic cells) for HL-60, TF-1 and THP-1 were 27 μM, 49 μM and 24 μM respectively. An excellent survival was seen on a KDR-negative cell line demonstrating that the cell death induced by PTK787/ZK 222584 is not a toxic effect. The primary blasts were overall 5–10 times more sensitive to PTK787/ZK 222584 than the cell lines, with a median LC50 value of 5.1 μM. Patient samples with a LC50 value below the median did not differ from patients above the median regarding age, sex, FAB classification or WBC count. With a response surface analysis we investigated the additional effect of PTK787/ZK 222584 upon cell death achieved by Amsacrine; we estimated the concentration combinations resulting in the same cell survival. We could show that, in cell lines as well as in primary AML blasts, the concentration of Amsacrine can be lowered and replaced for a certain dose of the potentially less toxic VEGFR inhibitor to achieve the same percentage of leukemic cell death. This study shows that PTK787/ZK 222584 might have more clinical potential in AML when combined with chemotherapy, e.g. Amsacrine. In addition, it is known from literature that topoisomerase inhibitors, e.g. Amsacrine, can cause treatment related AMLs. Moreover, many survivors of childhood or adolescent cancer experience treatment related cardiovascular complications that can impair their quality of life years after treatment. Therefore, reduction of complications and long-term effects of chemotherapy is warranted and might be achieved by lowering dosages of Amsacrine and replace it by other drugs with a lower toxicity profile, such as PTK787/ZK 222584.

Large Interindividual Differences in In Vitro Calicheamicin Sensitivity May Underly Gemtuzumab Ozogamicin Resistance in Acute Myeloid Leukemia (AML).

Gemtuzumab ozogamicin (GO or Mylotarg®) is increasingly being used in the treatment of AML. GO consists of a cytotoxic drug - calicheamicin which is conjugated to an anti-CD33 antibody. Most AML patients highly express CD33 on their blasts. Studies relating CD33 expression to response to GO have failed to show an association. Although 30–50% of patients respond to Mylotarg®, the causes of primary resistance to this drug remain unclear. Previous reports have studied P-gp status and CD33 expression as a cause of resistance to GO. Another factor that might determine response to GO is cellular drug resistance to calicheamicin.In this study we examined in vitro resistance to calicheamicin in 90 initially diagnosed and 32 relapsed pediatric AML samples using the 4 day MTT assay (concentration range 0.000004 – 0.4 μg/ml). The LC50 value, the drug concentration at which 50% of the cells is killed by the drug, is used as a measure of sensitivity. In addition to calicheamicin, some samples were also tested successfully for in vitro sensitivity to etoposide, cytarabine, daunorubicin, idarubicin, mitoxantrone, 6-thioguanine, vincristine and L-asparaginase.The characteristics of the 122 pediatric AML samples included are as follows: 62% boys, median age 9.6 years, median WBC 53.0*109/L, FAB types M0 n=9, M1 n=10, M2 n=18, M3 n=9, M4 n=33, M5 n=24, M7 n=3, unknown n=16. There was a more than 100,000 fold difference in calicheamicin sensitivity between the most sensitive and the most resistant patient samples. FAB M2 samples taken at initial diagnosis (n=13) were significantly more resistant to calicheamicin compared to the other FAB types (Resistance Ratio (RR)=2.5, median LC50 0.033 vs. 0.013 μg/ml, p=0.008). Newly diagnosed AML samples were significantly more sensitive to calicheamicin compared to relapsed AML samples (RR=0.68, median LC50 0.023 vs. 0.034 μg/ml, p=0.042) (although these patients had not been treated with calicheamicin). There was strong cross-resistance between calicheamicin and the anthracyclines idarubicin (Spearmans rho = 0.73, p<0.0001, n=23), daunorubicin (rho=0.61, p<0.0001, n=103) and mitoxantrone (rho=0.52, p=0.039, n=16). In addition, there was moderate cross-resistance with etoposide (rho=0.42, p<0.0001, n=101). No cross-resistance was observed between calicheamicin and cytarabine (rho=0.11, p=0.28, n=106), 6-thioguanine (rho=0.20, p=0.054, n=97), vincristine (rho=0.12, p=0.44, n=46) or L-asparaginase (rho=0.21, p=0.16, n=45). In conclusion, the interpatient differences in calicheamicin sensitivity are the largest differences in in vitro drug sensitivity we have ever observed in pediatric AML. FAB M2 samples are 2.5 fold more resistant to calicheamicin than samples with other FAB types. Initially diagnosed pediatric AML samples are 1.5 fold more sensitive to calicheamicin than relapsed AML samples. There is marked cross-resistance between calicheamicin and the related anthracyclin compounds. Given the large differences in sensitivity to calicheamicin in pediatric AML samples, it is likely that calicheamicin resistance plays a role in resistance to Mylotarg®.

In-Vitro Cytotoxicity of Tipifarnib (Zarnestra TM) in Pediatric AML and ALL Samples Is Independent of RAS Mutations.

The farnesyltransferase inhibitor tipifarnib (Zarnestra™) was originally developed to target malignancies harbouring RAS mutations. In the first clinical studies with tipifarnib, in adults with leukemia, it was found that patients who responded did not harbour any RAS mutations, suggesting a different mechanism of response. In a previous study we showed that 18% of 150 untreated pediatric AML patients harbour mutations in RAS, of which 30% were CBF-AML. We now studied 44 untreated and 13 relapsed pediatric AML, as well as 22 untreated ALL samples for mutations in RAS, using D-HPLC and direct sequencing. In vitro tipifarnib resistance was determined by a 4-day MTT assay (concentration 0.016-51μM, kindly provided by Janssen Research). The LC50 value, the concentration at which 50% of cells are killed by tipifarnib, was used as a measure of resistance. Patient characteristics were; for untreated AML: 64% boys; median age 9.3 years; median WBC 74.8x109/L; FAB 2xM0, 2xM1, 8xM2, 3xM3, 16xM4, 8xM5, 5x unclassified; for relapsed AML: 77% boys; median age 4.0 years; median WBC 41.6x109/L; FAB 2xM0, 2xM2, 3xM4, 2xM5, 2xM7, 2x unclassified; for untreated ALL: 73%boys; median age 6.0 years; median WBC 10.2x109/L; 15 B-cell precursor (BCP) ALL and 7 T-ALL.We found RAS mutations in 14 (32%) untreated AML samples (N-RAS : 8 samples exon 1, 1 sample exon 2; K-RAS: 5 samples exon 1 mutations). In relapsed AML 2 samples showed an N-RAS exon 1 mutation (15.4%). In ALL 18.2% had a RAS mutation: an N-RAS exon 1 mutation was found in 2 patients (9.1%) and a K-RAS exon 1 mutation in another 2 patients (9.1%). The distribution of tipifarnib sensitivity was similar in RAS mutated- and non-mutated untreated AML patients [median LC50 RAS mutated 7.1μM (P25-P75: 6.0-9.6μM) vs. non-mutated 4.9μM (P25-P75 2.3-8.2μM); p=0.199]. When we compared N-RAS mutated samples with K-RAS mutated samples there was no statistically significant difference in sensitivity to tipifarnib (median LC50 [p25-p75] 3.2μM [2.9-3.9μM] and 4.9μM [3.7-23.1μM], p=0.20), and comparing them separately with non-mutated AML did not show differences in sensitivity to tipifarnib (p=0.172 and p=0.463 respectively). One out of 9 (11%) N-RAS mutated and 3 out of 5 (60%) K-RAS mutated samples had an LC50 value above the 75th percentile for non-mutated AML and were considered resistant. Within relapsed AML the 2 RAS mutated samples had LC50 values of 0.83 and 6.3μM, versus a median value of 6.9μM for non-mutated relapsed AML. In ALL, we found similar results [median LC50 RAS mutated 7.8μM (P25-75: 4.1-12.8μM) vs. non-mutated 17.4μM (P25-75: 4.5-22.9μM), p=0.3], but the groups were very small.In conclusion, primary pediatric AML and ALL samples withRAS mutations show similar distributions of tipifarnib sensitivity as samples withoutRAS mutations. Hence, some RAS mutated samples may be relatively in-vitro resistant to tipifarnib, and some non-mutated samples may be relatively sensitive. Therefore, clinical studies with these compounds should not be restricted to RAS-mutated leukemia. Further studies are necessary to determine the molecular targets of farnesyltransferase inhibitors.

Post-induction residual leukemia in childhood acute lymphoblastic leukemia quantified by PCR correlates with in vitro prednisolone resistance

Most prognostic factors in childhood acute lymphoblastic leukemia (ALL) are informative for groups of patients, whereas new approaches are needed to predict the efficacy of chemotherapy for the individual patient. The residual leukemia following 4 weeks of induction therapy with prednisolone, vincristine, doxorubicin and i.t. methotrexate and the in vitro resistance to prednisolone, vincristine, and doxorubicin were measured in 30 boys and 12 girls with B (n = 34) or T lineage (n = 8) ALL. The residual leukemia was quantified after 2 (MRD-D15, n = 29) and 4 weeks (MRD-PI, n = 42) of induction therapy with a precise and reproducible clone-specific PCR technique. The median MRD-D15 and MRD-PI were 0.50% (75% range 0.0088.1%) and 0.014% (75% range 0.001-2.0%), respectively, and these levels correlated significantly (n = 29, rs = 0.75, P < 0.001). Both the MRD-D15 and the MRD-PI were related to the age of the patient (MRD-D15: rs= 0.48, P= 0.009; MRD-PI: rs = 0.45, P = 0.003). Patients with T lineage ALL had higher MRD-PI than those with B lineage ALL (median MRD-PI: 0.5% vs 0.01%, P = 0.05). The median LC50 (concentration lethal to 50% of cells) for prednisolone was 2.3 microg/ml (75% range 0.05-668). Both MRD-D15 and MRD-PI correlated significantly with the in vitro resistance to prednisolone (MRD-D15: rs = 0.41, P = 0.03; MRD-PI: rs = 0.39, P = 0.01); but not to in vitro vincristine or doxorubicin resistance. The correlations between MRD and in vitro prednisolone resistance were even more pronounced when B cell precursor and T cell leukemia were analyzed separately (B cell precursor ALL: MRD-PI vs prednisolone LC50: n = 33, rs = 0.47, P = 0.006; T cell ALL: MRD-PI vs prednisolone resistance: n = 8, rs = 0.84, P = 0.009). After a median follow-up of 5.0 years (75% range 3.2-6.9) eight patients have relapsed. All of the 21 patients with a MRD-PI < or =0.5% and a prednisolone LC50 < or =10 microg/ml have remained in remission whereas the 7 year event-free survival for the remaining 20 patients was 0.45 +/- 0.16 (P= 0.002) Prospective studies in childhood ALL are needed to clarify whether combined monitoring of in vitro drug resistance and residual leukemia early during chemotherapy could offer new ways to classify patients and stratify the intensity of therapy.

Leukaemia cell drug resistance and prognostic factors in AML.

In 93 cases of acute myeloid leukaemia (AML) the extent to which prognostic factors mirrored the in vitro cellular chemotherapy resistance (to anthracyclines aclarubicin (Acla) and daunorubicin (Dau) as well as nucleoside analogue cytarabine (Ara-C)) was investigated using a 4-d MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. We found that age at presentation and presence of secondary AML were significantly correlated to leukaemia cell Ara-C resistance. Thus, analysis of in vitro drug resistance data revealed that age at presentation and presence of secondary leukaemia were both independently correlated to cellular drug resistance, with older age being associated with higher Ara-C resistance in vitro (p=0.02 and 0.01 in univariate and multivariate analyses, respectively) and with secondary leukaemia being associated with higher Ara-C resistance (p=0.04 and 0.059 in univariate and multivariate analysis, respectively). Median LC-50 values (Ara-C) were: 178 ng/ml in paediatric cases, 356 ng/ml in younger adult cases, and 584 ng/ml in elderly (age > or = 60 yr) cases giving a resistance ratio between these age subgroups of 1:2.0:3.3. Median LC-50 values (Ara-C) was 381 ng/ml in de novo cases as opposed to 891 ng/ml (resistance ratio 1:2.3) in secondary cases. By contrast, cytogenetic findings, presenting leucocyte count, FAB-subtype, and gender were not consistently correlated to in vitro drug resistance to any of the three drugs. We conclude that at least two major adverse prognostic factors in AML (advanced age at presentation and presence of secondary leukaemia) are associated with increased leukaemia cell Ara-C resistance. High leucocyte count is not associated with increased cellular drug resistance towards Acla, Ara-C or Dau.

Comparison of the antileukemic activity in vitro of dexamethasone and prednisolone in childhood acute lymphoblastic leukemia

It is generally assumed that prednisolone (PRD) and dexamethasone (DXM) have equal glucocorticoid activity if PRD is given at sevenfold higher doses. Results of clinical studies of childhood acute lymphoblastic leukemia (ALL) suggested that DXM is more potent relative to PRD than assumed. The purpose of this study was to determine the relative antileukemic activity of PRD phosphate and DXM phosphate in 133 untreated childhood ALL samples in vitro, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) assay. There was a marked variation in antileukemic activity of both agents among the patient samples. The median LC50 (drug concentration lethal to 50% of the ALL cells) for PRD phosphate was 3.50 μM, for DXM phosphate 0.20 μM. The individually calculated ratios of the LC50 values for PRD and DXM phosphate showed a large range from 0.7 to >500, with a median of 16.2. This 16-fold difference could not be explained by differences between these glucocorticoids in stability, hydrolysis into unesterified drug, adhesion to the wall of the microculture plates, or protein binding. ALL cells were cross-resistant to PRD and DXM phosphate (correlation coefficient = 0.85, P < 0.000001). We conclude that the in vitro antileukemic activity of DXM phosphate is median 16-fold higher than that of PRD phosphate, which contrasts to the generally assumed factor of 7. Based on the higher potency of DXM, and its more favorable pharmacokinetics as reported in the literature, DXM may be preferred to PRD as the glucocorticoid in the treatment of ALL. © 1996 Wiley-Liss, Inc.

Natural Variability of Three Geographic Isolates of Gypsy Moth (Lepidoptera: Lymantriidae) Nuclear Polyhedrosis Virus

Variations in biological activities of three geographic isolates of gypsy moth, Lymantria dispar (L.), nuclear polyhedrosis virus (NPV) were determined in the laboratory. The median LC50 varied from 0.17 ± 0.19 polyhedral inclusion bodies (PIB) per mm2 (Abington, Massachusetts isolate) to 17.4 ± 13.5 PIB per mm2 (Dalmatia, Yugoslavia), a difference of about 102-fold. Activity of samples within the two North American isolates (Abington, Mass., and Hamden, Conn.) was approximately 15- and 20-fold, respectively; variation of individual samples within the Dalmatia isolate was approximately 17.5-fold at LC50. At LC90, variation was slightly greater within each North American isolate (23- and 35-fold for Abington and Hamden, respectively), but smaller for the Dalmatia isolate (14.5- fold). Samples of each NPV isolate exhibited a skewed distribution of activities at both LC50 and LC90. Plots of LC50 versus LC90 identified the samples within each isolate that merit selective propagation.

Reduced Growth of Coho Salmon Fry Exposed to Two Petroleum Components, Toluene and Naphthalene, in Fresh Water

Abstract Coho salmon, Oncorhynchus kisutch, fry were exposed for 40 days to stable, sublethal concentrations of toluene (0.4, 0.8, 1.6, 3.2, 5.8 μl/liter) and naphthalene (0.2, 0.4, 0.7, 1.4 mg/liter) in fresh water. All fry were fed equal daily rations of Oregon Moist Pellet Formula II. Dry weights, wet weights, and lengths of fry exposed to the two highest concentrations of each toxicant for 40 days were significantly less than controls (P < 0.01). Growth per day, determined from weights and lengths, decreased linearly with increased concentrations. Fry exposed to naphthalene had a slower growth rate than fry exposed to equivalent concentrations (percentage of the 96-hour median lethal concentration or LC50) of toluene. Concentrations 18% of the LC50 of naphthalene and 26% of the LC50 of toluene had no effect on dry weight, wet weight, or length of exposed fry.

TOXICITY OF BALLAST-WATER TREATMENT EFFLUENT TO MARINE ORGANISMS AT PORT VALDEZ, ALASKA*

ABSTRACT Approximately 12 million gallons of oily ballast water is taken ashore and treated daily at the Alyeska treatment plant, where tankers take on crude oil at the terminus of the Trans Alaska pipeline near Valdez, Alaska. Most oil is removed, but some light aromatic hydrocarbons (1 to 16 parts per million) remain in the large volume of discharged effluent. Between May and July, the concentration of aromatic hydrocarbons in the treated effluent (measured by gas chromatography) generally declined as the seasonal temperatures increased. We measured the toxicity of the effluent on site at Valdez. For the larvae of crustaceans and of fish the median lethal concentration LC50 was between 10 and 2p percent of treated effluent in 96-hour static tests. For salmon fry and shrimp in repeated acute flow-through assays, the (LC50) was quite consistent, between 20 and 40 percent of treated effluent. Because the concentration of aromatic hydrocarbons was much lower in the later tests but the toxicity of the effluent was not lower, toxicants other than aromatic hydrocarbons must contribute significantly to the toxicity of the effluent from the ballast-water treatment plant.