Arylamine N-acetyltransferases (NATs) were partially purified from human breast tumor tissues with complete separation of the isoforms in DEAE-Cellulose ion-exchange step. NAT with activity towards p-aminobenzoic acid (PABA) was isolated and purified from human breast tumor with 77 % yield and a purification factor of 5-fold. NAT with activity towards sulfamethazine (SMZ) was isolated and purified from human breast tumor with 21 % yield and a purification factor of 3-fold. Further purification attempts by Blue Sepharose affinity column chromatography resulted in the complete loss of both enzyme activities. The NAT1 purified from human breast tumor tissues had a molecular weight (Mr) value of about 27600 and an isoelectric point (pI) around 4.8, as confirmed by SDS-PAGE, IEF and Western blotting analysis. With immunohistochemical analysis, level of intensity of NAT1 immunostaining was observed to be going from weak in reduction mammoplasty samples to strongest in malignant breast tissue. The interindividual variation in the conjugation of p-aminobenzoic acid (PABA) and of sulfamethazine (SMZ) by cytosolic arylamine N-acetyltransferases (NATs) were investigated in 30 human breast tumor and matched samples. The average specific activity against PABA was calculated as 13&amp / #61617 / 2 pmole/min/mg protein for breast control NATs, and 20&amp / #61617 / 3 pmole/min/mg protein for breast tumor NATs. The average specific activity against SMZ was calculated as 12&amp / #61617 / 2 pmole/min/mg protein for breast control NATs, and 34&amp / #61617 / 6 pmole/min/mg protein for breast tumor NATs. Wilcoxon test revealed that the difference between the control and tumor groups is statistically significant with respect to the NAT1 activities as well as NAT2 activities. In three (3/30, 10%) patients tumor and tumor-free breast tissue NAT1 activity was not detectable. Among control tissues, the percentage of measurable NAT2 activity was 77% (23/30), while in tumor tissues it increased to 91%. Chemotherapy treatment was observed to have a slight inhibitory effect on mean NAT1 and NAT2 activities. There was an indication of a possible negative association with mean NAT1 activity and estrogen receptor status, while mean tumor NAT2 activity was observed to increase among estrogen receptor positive patients. Grade of malignancy seems to be positively associated with NAT1, but no such association could be suggested for NAT2 enzyme. Menopausal state of the patient was suggested to have a significant effect on NAT2 activity. Genotype determination of NATs revealed that NAT1*4 and NAT2*5A allele being most common among 10 breast cancer patients. NAT1*11 allele was prevalent among postmenopausal women. The putative rapid NAT1 genotypes was found to display lower control and tumor mean NAT1 activities compared to normal NAT1 genotypes. Among slow NAT2 acetylators, mean tumor NAT2 activities was found to be significantly higher than respective controls.

>Magister Scientiae - MSc / The human arylamine N-acetyltransferase 1 (NAT1) is critical in determining the duration of action and pharmacokinetics of amine-containing drugs such as para-aminosalicylic acid and para-aminobenzoyl glutamate used in clinical therapy of tuberculosis (TB), as well as influencing the balance between detoxification and metabolic activation of these drugs. SNPs in this enzyme are continuously being detected and indicate inter-ethnic and inter-individual variation in the enzyme function. The effect of nsSNPs on the structure and function of proteins are routinely analyzed using SIFT and POLYPHEN-2 prediction algorithms. The false-negative rate of these two algorithms results in as much as 25% of nsSNPs. This study aimed to explore the use of homology modeling including residue interactions, Gibbs free energy change and solvent accessibility as additional evidence for predicting nsSNP effects on enzyme function.This study evaluated the functional effects of 14 nsSNPs identified in a South African mixed ancestry population of which 3 nsSNPs were previously identified in Caucasians. The SNPs were evaluated using structural analysis that included homology modeling, residue interactions, relative solvent accessibility,Gibbs free energy change and sequence conservation in addition to the routinely used nsSNP function prediction algorithms, SIFT and POLYPHEN-2. The structural analysis implemented in this study showed a loss of hydrogen bonds for S259R thereby affecting protein function which contradicts predictions obtained from SIFT and POLYPHEN-2 algorithms. The variant N245I was shown to be neutral but contradicted the predictions from SIFT and POLYPHEN-2. Structural analysis predicted that variant R242M would affect protein stability and therefore NAT1 function in agreement with POLYPHEN-2 predictions but contradicting predictions from SIFT. No structural changes were expected for variant E264K in agreement with predictions obtained from POLYPHEN-2 but contradicting results from SIFT. The functions of the remaining 10 nsSNPs were consistent with those predicted by SIFT and POLYPHEN-2 namely that four variants R117T, E167Q, T193S and T240S do not affect the NAT1 function whereas R166T, F202V, Q210P, D229H, V231G and V235A could affect the enzyme function.This study provided the first evaluation of the functional effects of 11 newly characterized nsSNPs on the NAT1 tuberculosis drug-metabolizing enzyme. The six functionally important nsSNPs predicted by all three methods and the four SNPs with contradictory results will be tested experimentally by creating a SNP construct that will be cloned into an expression vector. These combined computational and experimental studies will advance our understanding of NAT1 structure-function relationships and allow us to interpret the NAT1 genetic polymorphisms in individuals who are slow or fast acetylators. The results, albeit a small dataset demonstrate that the routinely used algorithms are not without flaws and that improvements in functional prediction of nsSNPs can be obtained by close scrutiny of the molecular interactions of wild type and variant amino acids.

Aescin is a mixture of several acidic triterpenoid saponin glycosides found in the extracts of the horse chestnut tree. Horse chestnut, Aesculus Hipoocastanum, is one of the 25 domestic species of Aesculus that are mostly large, ornamental shade trees. Although known to be poisonous, the nuts of the horse chestnut are used by Amerindians, after detoxification. Horse chestnuts are said to have several traditional medicinal usages including even cancer. In this study the biochemical, genotoxic, and cytotoxic effects of aescin was studied using isolated lymphocytes, whole blood lymphocytes and HL-60 promyeloid leukemia cell lines. Cytotoxicity of aescin was examined by trypan blue viability staining of the cells in culture treated with varying aescin concentrations. It was observed that aescin was cytotoxic at all concentrations, for all cell types studied, except whole blood lymphocytes, where it was not cytotoxic at 10-9 and 10-10 M concentrations. Genotoxicity of aescin was examined by sister chromatid exchange and micronucleus. The genotoxic effect of Aescin was observed to be more significant over isolated lymphocytes compared to other cell lines. On the otherhand, aescin at 10-8 M and lower concentrations were observed to be non-genotoxic over whole blood lymphocytes whereas this concentration was considerably toxic for isolated lymphocytes and for HL-60 cell lines. Apoptotic properties of aescin were determined by DNA fragmentation, cytochrome c release and negative NAPO staining. All the Aescin concentrations tested resulted in apoptosis over HL-60 cell lines, whereas necrosis was not observed. However, isolated lymphocytes showed both apoptosis and necrosis upon treatment with 10-6 M to 10-8 M aescin, exhibiting apoptosis only at 10-9 M and 10-10 M. Biochemical effects of aescin were investigated by following GST and NAT enzyme activities. An increase in GST enzyme activity was observed over all cell lines treated with increasing aescin concentrations for 72 hours. Whereas NAT activity was decreased upon treatment with aescin in similar manner.