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Toxicity Analysis of Recombinant L-asparaginase I and II in Zebrafish.

ABSTRACT: L-asparaginases are extensively applied in the treatment of Acute Lymphoblastic Leukemia (ALL). The treatment regime of ALL consists of asparaginase from E. coli or Erwinia. The survival rate post-chemotherapy has increased to < 90% in recent years. Asparaginase therapy has also resulted in numerous toxicities to patients receiving therapy. This study demonstrates the reaction of normal cells of Danio rerio to asparaginase therapy. L-asparaginase I and II used in the present study are from two probiotic Lactobacillus species in comparison with a commercial L-asparaginase of E. coli origin. Zebrafish adults were injected with 2500 U/kg body weight of L-asparaginase treatments. The expression of SOD 2, CAT, GST, GTP BP3, FADS2 were analyzed with EF1α as house-keeping gene. The p value obtained proves that the data are significant. The histology of the L-asparaginase I treated fishes showed dilated sinusoids in the liver and pseudocyst in the pancreas. The L-asparaginase II and commercial asparaginase showed no pathology.

SUBMITTER: Suresh SA

PROVIDER: S-EPMC7539244 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Development of Processes for Recombinant L-Asparaginase II Production by Escherichia coli Bl21 (De3): From Shaker to Bioreactors.

Project description:Since 1961, L-asparaginase has been used to treat patients with acute lymphocytic leukemia. It rapidly depletes the plasma asparagine and deprives the blood cells of this circulating amino acid, essential for the metabolic cycles of cells. In the search for viable alternatives to produce L-asparaginase, this work aimed to produce this enzyme from Escherichia coli in a shaker and in a 3 L bioreactor. Three culture media were tested: defined, semi-defined and complex medium. L-asparaginase activity was quantified using the β-hydroxamate aspartic acid method. The defined medium provided the highest L-asparaginase activity. In induction studies, two inducers, lactose and its analog IPTG, were compared. Lactose was chosen as an inducer for the experiments conducted in the bioreactor due to its natural source, lower cost and lower toxicity. Batch and fed-batch cultures were carried out to reach high cell density and then start the induction. Batch cultivation provided a final cell concentration of 11 g L-1 and fed-batch cultivation produced 69.90 g L-1 of cells, which produced a volumetric activity of 43,954.79 U L-1 after lactose induction. L-asparaginase was produced in a shaker and scaled up to a bioreactor, increasing 23-fold the cell concentration and thus, the enzyme productivity.

| S-EPMC7823503 | biostudies-literature

Asparaginase-associated toxicity in children with acute lymphoblastic leukemia.

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| S-EPMC4819847 | biostudies-literature

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| S-EPMC6896745 | biostudies-literature

l-Asparaginase Type II from Fusarium proliferatum: Heterologous Expression and In Silico Analysis.

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| S-EPMC10534586 | biostudies-literature

Activity and Toxicity of Intravenous Erwinia Asparaginase Following Allergy to E. coli-Derived Asparaginase in Children and Adolescents With Acute Lymphoblastic Leukemia.

Project description:BackgroundErwinia asparaginase is antigenically distinct from E.coli-derived asparaginase and may be used after E.coli-derived asparaginase hypersensitivity. In a single-arm, multicenter study, we evaluated nadir serum asparaginase activity (NSAA) and toxicity with intravenously administered asparaginase Erwinia chrysanthemi (IV-Erwinia) in children and adolescents with acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma with hypersensitivity to E.coli-derived asparaginase.Patients and methodsBetween 2012 and 2013, 30 patients (age 1-17 years) enrolled from 10 centers. Patients received IV-Erwinia, 25,000 IU/m(2)/dose on Monday/Wednesday/Friday, for 2 consecutive-weeks (6 doses = 1 cycle) for each dose of pegaspargase remaining in the original treatment plan. The primary objective was to determine the proportion of patients achieving NSAA ≥ 0.1 IU/ml 48 hr after dose 5 in Cycle 1. Secondary objectives included determining the proportion achieving NSAA ≥ 0.1 IU/ml 72 hr after Cycle 1 dose 6, and the frequency of asparaginase-related toxicities.ResultsTwenty-six patients completed Cycle 1; 24 were evaluable for NSAA assessment. In Cycle 1, NSAA ≥ 0.10 IU/ml was detected in 83% of patients (95% confidence interval [CI], 63-95%) 48 hr post-dose 5 (mean ± SD; 0.32 IU/ml ± 0.23), and in 43% (95% CI, 22-66%) 72 hr post-dose 6 (mean ± SD; 0.089 IU/ml ± 0.072). For all 30 patients over all cycles, hypersensitivity/infusional reactions with IV-Erwinia occurred in 37%, pancreatitis 7%, and thrombosis 3%.ConclusionsIV-Erwinia administration in children/adolescents appeared feasible and tolerable. A therapeutically-effective NSAA (≥ 0.10 IU/ml) was achieved in most patients at 48 hr, but in fewer than half 72 hr post-dosing, suggesting that monitoring NSAA levels and/or every 48 hr dosing may be indicated.

| S-EPMC4715717 | biostudies-literature

Fed-Batch Production of Saccharomyces cerevisiae L-Asparaginase II by Recombinant Pichia pastoris MUT s Strain.

Project description:L-Asparaginase (ASNase) is used in the treatment of acute lymphoblastic leukemia, being produced and commercialized only from bacterial sources. Alternative Saccharomyces cerevisiae ASNase II coded by the ASP3 gene was biosynthesized by recombinant Pichia pastoris MUT s under the control of the AOX1 promoter, using different cultivation strategies. In particular, we applied multistage fed-batch cultivation divided in four distinct phases to produce ASNase II and determine the fermentation parameters, namely specific growth rate, biomass yield, and enzyme activity. Cultivation of recombinant P. pastoris under favorable conditions in a modified defined medium ensured a dry biomass concentration of 31 gdcw.L-1 during glycerol batch phase, corresponding to a biomass yield of 0.77 gdcw.g glycerol-1 and a specific growth rate of 0.21 h-1. After 12 h of glycerol feeding under limiting conditions, cell concentration achieved 65 gdcw.L-1 while ethanol concentration was very low. During the phase of methanol induction, biomass concentration achieved 91 gdcw.L-1, periplasmic specific enzyme activity 37.1 U.g dcw-1 , volumetric enzyme activity 3,315 U.L-1, overall enzyme volumetric productivity 31 U.L-1.h-1, while the specific growth rate fell to 0.039 h-1. Our results showed that the best strategy employed for the ASNase II production was using glycerol fed-batch phase with pseudo exponential feeding plus induction with continuous methanol feeding.

| S-EPMC6375902 | biostudies-literature

Anticancer Activity of Extremely Effective Recombinant L-Asparaginase from Burkholderia pseudomallei.

Project description:L-asparaginase (E.C. 3.5.1.1) purified from bacterial cells is widely used in the food industry, as well as in the treatment of childhood acute lymphoblastic leukemia. In the present study, the Burkholderia pseudomallei L-asparaginase gene was cloned into the pGEX-2T DNA plasmid, expressed in E. coli BL21 (DE3) pLysS, and purified to homogeneity using Glutathione Sepharose chromatography with 7.26 purification fold and 16.01% recovery. The purified enzyme exhibited a molecular weight of ~33.6 kDa with SDS-PAGE and showed maximal activity at 50°C and pH 8.0. It retained 95.1, 89.6%, and 70.2% initial activity after 60 min at 30°C, 40°C, and 50°C, respectively. The enzyme reserved its activity at 30°C and 37°C up to 24 h. The enzyme had optimum pH of 8 and reserved 50% activity up to 24 h. The recombinant enzyme showed the highest substrate specificity towards L-asparaginase substrate, while no detectable specificity was observed for L-glutamine, urea, and acrylamide at 10 mM concentration. THP-1, a human leukemia cell line, displayed significant morphological alterations after being treated with recombinant L-asparaginase and the IC50 of the purified enzyme was recorded as 0.8 IU. Furthermore, the purified recombinant L-asparaginase improved cytotoxicity in liver cancer HepG2 and breast cancer MCF-7 cell lines, with IC50 values of 1.53 and 18 IU, respectively.

| S-EPMC9628870 | biostudies-literature

Recombinant L-asparaginase 1 from Saccharomyces cerevisiae: an allosteric enzyme with antineoplastic activity.

Project description:L-asparaginase (L-ASNase) (EC 3.5.1.1) is an important enzyme for the treatment of acute lymphoblastic leukaemia. Currently, the enzyme is obtained from bacteria, Escherichia coli and Erwinia chrysanthemi. The bacterial enzymes family is subdivided in type I and type II; nevertheless, only type II have been employed in therapeutic proceedings. However, bacterial enzymes are susceptible to induce immune responses, leading to a high incidence of adverse effects compromising the effectiveness of the treatment. Therefore, alternative sources of L-ASNase may be useful to reduce toxicity and enhance efficacy. The yeast Saccharomyces cerevisiae has the ASP1 gene responsible for encoding L-asparaginase 1 (ScASNase1), an enzyme predicted as type II, like bacterial therapeutic isoforms, but it has been poorly studied. Here we characterised ScASNase1 using a recombinant enzyme purified by affinity chromatography. ScASNase1 has specific activity of 196.2 U/mg and allosteric behaviour, like type I enzymes, but with a low K0.5 = 75 μM like therapeutic type II. We showed through site-directed mutagenesis that the T64-Y78-T141-K215 residues are involved in catalysis. Furthermore, ScASNase1 showed cytotoxicity for the MOLT-4 leukemic cell lineage. Our data show that ScASNase1 has characteristics described for the two subfamilies of l-asparaginase, types I and II, and may have promising antineoplastic properties.

| S-EPMC5099943 | biostudies-literature

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