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Expression and characterization of human lactoperoxidase single nucleotide polymorphisms in an insect cell system
No full textHuman lactoperoxidase (LPO) is an enzyme that catalyzes the hydrogen peroxide-mediated oxidation of (pseudo)halides and organic substrates. Its role is as part of the antimicrobial defence system, within the secretions and on the mucosal surfaces where it occurs. LPO is also able to catalyze the oxidation of a wide variety of xenobiotics and endogenous compounds. Recently several single nucleotide polymorphisms (SNPs) of LPO have been identified, and three were investigated in the present study. The variants R414Q, V421M, and R514Q were expressed as C-terminally his-tagged proteins using a plasmid mediated insect cell expression system and purified by affinity chromatography. Recombinant LPO was obtained for all three SNP variants and wild type. Wild type LPO and variant R514Q displayed characteristic LPO absorbance spectra and similar specific activities with ABTS (2,2'-azinobis-[3-ethylbenzthiazoline-6-sulphonic acid]) as substrate. However, variants R414Q and V421M showed no characteristic LPO spectra or peroxidase activity. Results suggest that these variants fail to acquire the heme prosthetic group during biosynthesis due to improper protein folding or due to disruption ofthe local protein environment, which impedes heme incorporation to a significant level. These findings suggest that expression of LPO holoenzyme is sensitive to mutation at these positions and individuals who carry these SNPS may show a deficiency with regard to their expression of active LPO
Expression of recombinant human cytochrome P450 in Ames test strains for mutagenicity testing
No full textHeterologous expression of mammalian bioactivation enzymes in bacteria can obviate the requirement for the addition of exogenous enzymes in bacterial genotoxicity testing. Ames tester strains bearing a plasmid which carries the cDNA of human P450 1A1 were constructed. It was determined that both NADPH-cytochrome P450 reductase and plasmid pKM101, which carries the genes for error-prone DNA repair (mucAB), were likely required for the activation of P450 1A1-dependent mutagens. Subsequently, tester strains bearing plasmids coding for a P450 1A1:NADPH-cytochrome P450 reductase fusion protein and the MucAB proteins were constructed. Strain testing indicated that functional MucAB proteins were produced. However, functional P450 1A1 was not detected in any of the strains. Immunoblot analysis indicated that the level of P450 1A1 expression was low in strains bearing both plasmids. The presence of the plasmid bearing the mucAB genes may have affected the level of P450 1A1 expression
Affinity purification of porcine kidney enzymes that bind mercapturic acids
Get PDFMercapturic acid (N-acetylcysteine S-conjugate) formation is a major route for the metabolism and elimination of the glutathione conjugates formed from endogenous and exogenous compounds, but the enzymes of the mercapturic acid pathway are still not completely characterized. In the present study, an affinity purification strategy was developed for identifying enzymes that act on mercapturic acids. Mono- and di-mercapturates of dopamine were synthesized, purified by HPLC, and covalently coupled to an agarose column. Bound proteins were eluted with a dopamine mercapturate, trypsinized, and identified by LC-MS-MS. Identified proteins included four dipeptidase enzymes; angiotensin converting enzyme, dipeptidyl-peptidase IV, membrane dipeptidase and aminopeptidase N. These dipeptidases were tested for their role in the hydrolysis of cysteinylglycine S-conjugates in the mercapturic acid pathway. Results from this study suggest that aminopeptidase and membrane dipeptidase may be involved in the hydrolysis of cysteinylglycine S-conjugates, while angiotensin converting enzyme does not contribute to the pathway
Analysis of heterocyclic aromatic amines in human breast milk
No full textThis thesis describes a new solid phase extraction procedure that was developed for the detection of 2-amino-1-methyl-6-phenylimidazo[4,5-' b']pyridine (PhIP) and other heterocyclic aromatic amines in human breast milk. Analysis was carried out with high-performance liquid chromatography/electrospray ionization/tandem mass spectrometry (HPLC/ESI-MS/MS) with selective reaction monitoring detection. The use of this analysis method allowed PhIP to be quantitated at levels as low as 0.7 pg/mL. The level of PhIP in human breast milk was found to be much lower than previously published; it was above the limit of quantification in only one of 48 samples. That sample had a PhIP concentration of 1.0 pg/mL. A time course study was also carried out comparing the level of PhIP in breast milk to the time of consumption of a well-cooked chicken breast. Heterocyclic aromatic amines were present in breast milk approximately 20 hours after consumption of a well-done chicken meal
A random mutagenesis approach to the construction and characterization of human N-acetyltransferase variants
No full textAcetyl CoA: arylamine 'N'-acetyltransferase (NAT) enzymes catalyze the 'N'-acetylation of aromatic amines and the 'O'-acetylation of aryl hydroxylamines, reactions which govern the disposition and toxicity of many drugs and carcinogens. The human NAT genes and enzymes NAT1 and NAT2 are highly polymorphic and constitute one of the best-studied examples of the genetic control of drug metabolism. Naturally-occurring human NAT variants provide limited insight into the relationship between NAT amino acid sequence and enzyme activity. We have shown previously that the expression of recombinant NAT2 in bacterial tester strains results in greatly enhanced sensitivity to mutagenic nitroaromatic compounds (which are reduced to aryl hydroxylamines by bacterial enzymes). We hypothesized that random mutagenesis combined with rapid screening could be used to identify functionally-significant amino acid residues in NAT enzymes. Pools of NAT2 variants were generated by PCR-mediated random mutagenesis of the complete coding sequence. Reversion induced by an NAT-dependent mutagen, nitro-IQ, was used as the basis for screening these pools to identify variants with altered enzyme activity. Eighteen variants were characterized by quantitative mutagenicity assays and enzyme kinetic measurements. This approach can provide new insight into the biochemistry of enzymes involved in the metabolic activation of mutagens
The role of residue M232 in the C-terminal helix of human glutathione transferase T1-1
Get PDFGlutathione transferases (GSTs) catalyze the glutathione (GSH) conjugation of various xenobiotics and endogenous compounds. GSTs play a pivotal role in cellular defence, as they are the main contributors to the inactivation of genotoxic compounds. A primary determinant for divergent substrate acceptance among GSTs appears to be the electrophilic binding site or “H-site”. Shokeer et al. (2010) reported that replacement of residue methionine 232 by alanine in mouse GST T1-1 greatly altered the enzyme’s catalytic activities. The corresponding residue in human enzyme is also methionine, but, in other mammalian GST T1-1 orthologues, arginine (horse; Equus caballus) or leucine (pig; Sus scrofa) residues are found at this position. The goal of the present study aims to investigate the role of residue 232 (located in the C-terminal α-helix) in the structure and function of human GST T1-1 protein. Three variants (M232R, M232L and M232W) were constructed and expressed in Escherichia coli. The effects of these mutations on protein expression, catalytic activity and structure were tested. Variants M232R and M232L results were generally unremarkable, showing only minor differences from the wild-type protein. However, variant M232W showed significant decreases in protein expression, had reduced specific activities and showed substantial changes in the CD spectrum. The spectral shift was consistent with the interpretation that the C-terminal α-helix unwinds into a random-coil conformation. Unlike horse and pig, no mammalian species is known to have a GST T1-1 enzyme with a tryptophan residue at position 232. Results from this study shows that residue 232 does not critically affect the structure and function of human GST T1-1
Analysis of human breast milk for the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine
No full textThe heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-' b']pyridine (PhIP) is found in meats prepared at ordinary cooking temperatures. PhIP is a probable human carcinogen that is implicated in the increased incidence of breast cancer. In a previous study (DeBruin, L. S., ' et al.' (2001) 'Chem. Res. Toxicol.' 14: 1523-1528), PhIP was found to be present in the milk of nine women who ate meat as a regular part of their diet. Milk samples were collected from two studies: a comparison of PhIP levels in the milk of vegetarian and non-vegetarian women, and a dietary intervention study to observe the changing levels of PhIP in breast milk following a meal of well-cooked chicken. In the samples analyzed, it was not possible to quantify or detect PhIP, due to the presence however, further development is required to obtain a method suitable for the analysis of a large sample series
Lidocaine and 2,6-dimethylaniline in bovine and human milk
No full text2,6-Dimethylaniline (2,6-DMA) is an aromatic amine which can be produced by metabolism of the local anaesthetic lidocaine. 2,6-DMA is also found in cigarette smoke. While 2,6-DMA is a moderate carcinogen in rats, its carcinogenicity in humans is unknown. The purpose of this research was to determine whether 2,6-DMA is transferrable to human and bovine milk. Therefore, milk samples were obtained from bovines treated with lidocaine (2.9-3.9 mg/kg). A breast milk sample was also obtained from a donor who had previously received 36 mg lidocaine for a dental procedure. In addition, milk from smokers and non-smokers was analysed. The method of solid phase microextraction (SPME) coupled with gas chromatography/mass spectrometry (GC/MS) was optimized and used for the analysis of 2,6-DMA in milk. Lidocaine concentrations were also determined by an ELISA assay. Upon analysis of the milk samples obtained as described above, 2,6-DMA was found at a level of 1.60 ppb in human breast milk. 2,6-DMA was also present at levels ranging from 14.5-66.0 ppb in bovine milk as described above. 2,6-DMA was present at a level of 1.31 ppb in the milk of one smoker (n = 4), while it was not detected in the milk of non-smokers (n = 15). These results show that 2,6-DMA is transferrable to both bovine and human milk after individuals are injected with lidocaine, and possibly after exposure to cigarette smoke
Characterization and functional analysis of human cytochrome P450 1A2 variants expressed in Escherichia coli
No full textCytochrome P450s are a superfamily of enzymes that participate in the metabolism of various xenobiotics and endogenous compounds. An increasing number of studies demonstrate that human P450 enzymes are highly polymorphic. In 2001, Chevalier 'et al.' reported four human 1A2 allelic variants (D348N, 1386F, C406Y and R431W). The present study was to investigate the effects of these mutations on the enzyme expression and activity. Four variants displayed different expression of holoenzymes in 'Escherichia coli': compared with wild type, expression was slightly lower in C406Y, less than half in D348N and I386F, and undetectable in variant R431W. Activities for the activation of procarcinogens MeIQ (2-amino-2,4-dimethyl-imidazo[4,5-' f']quinoline) and IQ (2-amino-3-methylimidazo[4,5-'f']quinoline) were roughly agreed with their expression levels, with complete loss of catalytic ability in variant R431W. The values of 'kcat/K' M of 'N'-hydroxylation of five aromatic amines and ' O'-deethylation of phenacetin varied from 0.1- to 3.0-fold of the wild type. Results from this study show that the residue R431, located in the "meander" region, is critical for human P450 1A2
An in vitro assay of chemical mutagenicity in rat mammary epithelial and fibroblast cells
No full textEpithelial cells are the primary site for carcinogenesis in most tissues, while stromal cells rarely develop into tumours. To investigate the basis of this differential susceptibility, three cell lines were developed from the oral epithelium (BBR/OE), mammary epithelium (BBR/ME), and stromal mammary fibroblasts (BBR/MFib) of transgenc Big Blue rats. These animals carry multiple chromosomal copies of a bacteriophage transgene for mutant frequency analysis using a selective assay. The different cell types were characterized cytogenetically and tested for chemically-induced cytotoxicity and for genotoxicity in the transgene ('cII'). The metabolic basis for their relative responses was investigated. For the direct-acting mutagen 'N'-ethyl-' N'-nitrosourea, all cell lines were equally sensitive to mutagenesis in the 'cII' gene but the fibroblast cell line showed greater resistance to cytotoxicity. For the dietary mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-' b']pyridine (PhIP), rat liver S9 was used to supply cytochrome P450 and conjugating enzymes. Mutagenicity was observed in the fibroblast cells, but the greater toxicity of PhIP to the epithelial cells prevented a definitive evaluation of mutagenicity. When the cells were treated with N-OH-PbIP, the epithelial cells were again more sensitive to cytotoxicity but a small increase in mutagenicity was observed for both mammary cell lines. RT-PCR analysis showed that cytochrome P450 enzymes 1A1 and 1B1 are expressed at low levels in both cell lines, with greater 1B1 expression seen in the fibroblast cells. In contrast, greater 'O'-acetyltransferase expression was measured in the mammary epithelial cells. It can be concluded that insufficient P450 activity is expressed to produce the hydroxylated metabolite of PhIP and that other conjugating enzymes may play a role in producing the ultimate mutagen. For the combustion products benzo['a']pyrene (B['a']P) and dimethyl-benz['a']anthracene (DMBA), a strong cytotoxicity and mutagenicity response was observed only in the fibroblast cells. Furthermore, this response occurred in the absence of exogenous bioactivation and could be reduced, in a dose-dependent fashion, by the P450 1A1 and 1B1 inhibitor [alpha]-naphthoflavone. Continued work with these cell lines may provide further insight into the role that chemical mutagens play in carcinogenesis in specific cell populations
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