| Classical phenylketonuria (PKU) is a rare metabolic disorder that usually results from a deficiency of a liver enzyme known as phenylalanine hydroxylase (PAH). This enzyme deficiency leads to elevated levels of the phenylalanine (Phe) in the blood and other tissues, mental retardation, microcephaly, delayed speech, seizures, eczema, behavior abnormalities, and other symptoms characterize the untreated state. Present treatment relies on the observation that a (semisynthetic) diet low in phenylalanine will prevent HPA and thus the disease. Because it involves a major alteration of lifestyle, dietary treatment is difficult, especially; Surveys of clinical practices suggest that most clinics advocate lifelong dietary treatment for metabolic control of blood Phe levels. Other therapy such as somatic gene therapy, oral enzyme therapy with phenylalanine ammonia lyase (PAL) and so on has been recommended.Namely. A combination of oral enzyme therapy with PAL (EC 4.3.1.5) and controlled low protein diet might replace dependence on the semisynthetic diet, for treatment of PKU after infancy. PAL is a robust autocatalytic enzyme that converts phenylalanine to metabolically insignificant amounts of ammonia and trans-cinnamic acid, a harmless metabolite; the later is converted to benzoic acid and rapidly excreted in urine as hippurate. A preliminary report indicates that HPA is attenuated by oral administration of microencapsulated PAL in the rat with chemically induced HPA and also, in preliminary studies, in naturally occurring HPA in amouse model. Preliminary studies in human PKU patients showed analogous .responses after the administration of PAL in entericcoated gelatin capsules or during use of an extracorporeal enzyme reactor. The human and even the animal studies were not continued because PAL was not available in sufficient amounts at reasonable cost.Over the past few years, others and Cai et al in our laboratory had successfully used a cDNA of the rice PAL gene, under the control of a high-expression promoter T7 and expressed it in a strain of E coli BL21DE3 to obtain large amounts of PAL. The engineering bacteria named BL2 lDE3pET28C-rm-1-cDNA were stored in -70'C. Induced by 1PTG and disrupted by sonic, the crude extracts possess PAL bioactivities, which can be used to reduce [Phe] in the blood.It is necessary to test its genotoxicity for BL21DE3pET28c-rPAL-1-cDNA extract being used as drug. According to genotoxicologic guidelines of ICH and IWGTP (international workshop on genotoxicity test procedures), in conjunction with conditions of our laboratory, Ames test, in vitro cytokinesis-block micronucleus (CBMN) assay and single cell gel electrophoresis (SCGE) was selected.The extract of expressed E.coli BL21DE3pET28c-rPAL-1-cDNA, BL2lDE3pET28c induced by IPTG and cinnamic acid with unknown genotoxic potential were test comparatively in Ames test (using TA97, TA98, TA100 and TAI02), SCGE and CBMN (using FL cells). The comparison of results is generally based on at least three independent experiments, each with two replicate cultures at a minimum of three concentrations. A high degree of concordance between results of Ames test, SCGE and CBMN was observed. CINN has no mutagenic activities with or without S9 in Ames test, as well as in SCGE and CBMN. Whereas the extract of expressed E.coli BL21DE3pET28c-rpA1-1-cDNA, BL21DE3pET28c led to increased numbers of revertant with or without S9 in Ames test, and at some concentration, the revertant numbers were over twofold in comparison with negative control in every tester strains. In CBMN, the extract of expressed E.coli BL21DE3pET28c-rPAL-1-cDNA, BL21DE3pET28c engendered elevated MN cell rate using P-NF induced FL cells. Above the concentration 0.5mg/ml, the elevated MN cell rate wereNF induced FL cells. Above the concentration 0.5mg/ml, the elevated MN cell rate were significant compared to negative control, P<0.05 or 0.01. However, similar phenomena were not observed using FL cells directly. In the SCGE, the average comet length of 0.05mg/ml the extra...
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