A 3-methylcrotonyl-CoA carboxylase deficient human skin fibroblast transcriptome reveals underlying mitochondrial dysfunction and oxidative stress.

Isolated 3-methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive inherited metabolic disease of leucine catabolism with a highly variable phenotype. Apart from extensive mutation analyses of the MCCC1 and MCCC2 genes encoding 3-methylcrotonyl-CoA carboxylase (EC 6.4.1.4), molecular data on MCC deficiency gene expression studies in human tissues is lacking. For IEMs, unbiased '-omics' approaches are starting to reveal the secondary cellular responses to defects in biochemical pathways. Here we present the first whole genome expression profile of immortalized cultured skin fibroblast cells of two clinically affected MCC deficient patients and two healthy individuals generated using Affymetrix(®)HuExST1.0 arrays. There were 16191 significantly differentially expressed transcript IDs of which 3591 were well annotated and present in the predefined knowledge database of Ingenuity Pathway Analysis software used for downstream functional analyses. The most noticeable feature of this MCCA deficient skin fibroblast transcriptome was the typical genetic hallmark of mitochondrial dysfunction, decreased antioxidant response and disruption of energy homeostasis, which was confirmed by mitochondrial functional analyses. The MCC deficient transcriptome seems to predict oxidative stress that could alter the complex secondary cellular response that involve genes of the glycolysis, the TCA cycle, OXPHOS, gluconeogenesis, ß-oxidation and the branched-chain fatty acid metabolism. An important emerging insight from this human MCCA transcriptome in combination with previous reports is that chronic exposure to the primary and secondary metabolites of MCC deficiency and the resulting oxidative stress might impact adversely on the quality of life and energy levels, irrespective of whether MCC deficient individuals are clinically affected or asymptomatic.

Leukocyte telomere length and hemostatic factors in a South African cohort: the SABPA Study.

BACKGROUND: Incident atherothrombotic disease is predicted by leukocyte telomere length, a marker of biological age, and hemostatic factor levels, indicating a hypercoagulable state. We hypothesized that shorter telomeres are associated with elevated circulating levels of hemostatic factors. METHODS: We examined 171 South African (black) and 182 Caucasian (white) schoolteachers (mean age ± standard deviation, 48.5 ± 9.0 years; 50.4% women). Levels of fibrinogen, von Willebrand factor antigen (VWF:Ag), D-dimer and plasminogen activator inhibitor-1 antigen (PAI-1:Ag) were measured in plasma, and values were log-transformed before analysis. Relative average telomere length (content of telomere PCR product/content of human ß-globin PCR product ratio, i.e. telomere/single-copy gene ratio) was assessed with multiplex quantitative real-time PCRs. Multivariate analyses included demographics, metabolic factors, health behavior, and medication. RESULTS: Africans had shorter mean telomere length (0.82, 95% confidence interval [CI] 0.79-0.86 vs. 1.07, 95% CI 1.04-1.10) and higher fibrinogen (B = 0.085, 95% CI 0.061-0.109) and PAI-1:Ag (B = 0.255, 95% CI 0.206-0.303) levels, but lower VWF:Ag levels (B = - 0.059, 95% CI - 0.089 to - 0.028), than Caucasians. Shorter telomeres were associated with higher fibrinogen (B = - 0.045, 95% CI - 0.088 to - 0.001), VWF:Ag (B = - 0.137, 95% CI - 0.193 to - 0.081) and D-dimer (B = - 0.201, 95% CI - 0.377 to - 0.025) levels, conditional on ethnicity. An interaction emerged between ethnicity and telomere length for VWF:Ag level; that is, shorter telomeres were associated with higher VWF:Ag levels in Caucasians (B = - 0.170, 95% CI - 0.232 to - 0.108) but not in Africans. CONCLUSIONS: Shorter telomeres were associated with increased levels of several hemostatic factors after adjustment for confounding variables, whereby ethnicity partially moderated this effect. A relationship between accelerated biological aging and hypercoagulability might contribute to the risk of premature atherothrombotic events.

8-Oxo-7,8-dihydro-2'-deoxyguanosine, reactive oxygen species and ambulatory blood pressure in African and Caucasian men: the SABPA study.

Various studies indicate a relationship between increased oxidative stress and hypertension, resulting in increased DNA damage and consequent excretion of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). The aim of this study was to compare urinary 8-oxodG levels in African and Caucasian men and to investigate the association between ambulatory blood pressure (BP) and pulse pressure (PP) with 8-oxodG in these groups. We included 98 African and 92 Caucasian men in the study and determined their ambulatory BP and PP. Biochemical analyses included, urinary 8-oxodG, reactive oxygen species (ROS) (measured as serum peroxides), ferric reducing antioxidant power (FRAP), total glutathione (GSH), glutathione peroxidase (GPx) and glutathione reductase (GR) activity. The African men had significantly higher systolic (SBP) and diastolic blood pressure (DBP) (both p < 0.001). Assessment of the oxidative stress markers indicated significantly lower 8-oxodG levels (p < 0.001) in the African group. The African men also had significantly higher ROS (p = 0.002) with concomitant lower FRAP (p < 0.001), while their GSH levels (p = 0.013) and GR activity (p < 0.001) were significantly higher. Single and partial regression analyses indicated a negative association between urinary 8-oxodG levels with SBP, DBP and PP only in African men. These associations were confirmed in multiple regression analyses (SBP: R(2) = 0.41; ß = -0.25; p = 0.002, DBP: R(2) = 0.30; ß = -0.21; p = 0.022, PP: R(2) = 0.30; ß = -0.19; p = 0.03). Our results revealed significantly lower urinary 8-oxodG in African men, accompanied by a negative association with BP and PP. We propose that this may indicate a dose-response relationship in which increased oxidative stress may play a central role in the up-regulation of antioxidant defence and DNA repair mechanisms.

Inhibition of N-acetylglutamate synthase by various monocarboxylic and dicarboxylic short-chain coenzyme A esters and the production of alternative glutamate esters.

Hyperammonemia is a frequent finding in various organic acidemias. One possible mechanism involves the inhibition of the enzyme N-acetylglutamate synthase (NAGS), by short-chain acyl-CoAs which accumulate due to defective catabolism of amino acids and/or fatty acids in the cell. The aim of this study was to investigate the effect of various acyl-CoAs on the activity of NAGS in conjunction with the formation of glutamate esters. NAGS activity was measured in vitro using a sensitive enzyme assay with ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) product analysis. Propionyl-CoA and butyryl-CoA proved to be the most powerful inhibitors of N-acetylglutamate (NAG) formation. Branched-chain amino acid related CoAs (isovaleryl-CoA, 3-methylcrotonyl-CoA, isobutyryl-CoA) showed less pronounced inhibition of NAGS whereas the dicarboxylic short-chain acyl-CoAs (methylmalonyl-CoA, succinyl-CoA, glutaryl-CoA) had the least inhibitory effect. Subsequent work showed that the most powerful inhibitors also proved to be the best substrates in the formation of N-acylglutamates. Furthermore, we identified N-isovalerylglutamate, N-3-methylcrotonylglutamate and N-isobutyrylglutamate (the latter two in trace amounts), in the urines of patients with different organic acidemias. Collectively, these findings explain one of the contributing factors to secondary hyperammonemia, which lead to the reduced in vivo flux through the urea cycle in organic acidemias and result in the inadequate elimination of ammonia.

Investigating the effects of the presence of foreign DNA on DNA methylation and DNA repair events in cultured eukaryotic cells.

Methylation of DNA in eukaryotic cells, global as well as gene-specific, is affected by endogenous and endogenous factors. In this paper, it is reported that deviations in DNA methylation and expression of genes involved in DNA repair and the cell cycle are affected in 143B cultured cells containing an expression vector. Global DNA methylation analysis with cytosine-extension assay revealed a decreased global DNA methylation in the presence of the expression vector. Less promoter-specific methylation, as measured by bisulfite-MS PCR, was observed for MGMT and p16INK4a in vector-containing cells. Comet assay investigations revealed a negative effect on the DNA repair capacity of both BER and NER in Complex III compromised cells. This was reflected in the down-regulation of hOGG1 and ERCC1 expression. The results presented in this paper support the existence of a strong relationship between impaired mitochondrial function and deviations in DNA methylation and extend this relationship to impaired DNA repair.

Associations between reactive oxygen species, blood pressure and arterial stiffness in black South Africans: the SABPA study.

Many mechanisms, including oxidative stress, contribute to hypertension. This study investigated the possible associations between oxidative stress, blood pressure and arterial stiffness in black South Africans. Ambulatory blood pressure measurements were taken for 101 black South African men and 99 women. The stiffness indices included ambulatory arterial stiffness index (AASI) and pulse pressure (PP). Reactive oxygen species (ROS) levels (P<0.0001) were higher in the African women compared with men. ROS levels were also higher in hypertensive compared with normotensive men. The 24 h systolic blood pressure (SBP; P<0.01), 24 h diastolic blood pressure (DBP; P<0.0001) and pulse wave velocity (PWV; P<0.01) were significantly higher in African men compared with women. There were unadjusted positive associations of 24 h SBP (r=0.33; P=0.001), 24 h DBP (r=0.26; P=0.008) and 24 h PP (r=0.29; P=0.003) with ROS in African men only. A positive association between AASI and ROS existed only in hypertensive men (r=0.27; P=0.035), but became nonsignificant (B=0.0014; P=0.14) after adjustments. Adjusted, positive associations of 24 h SBP (B=0.181; P=0.018) and 24 h PP (B=0.086; P=0.050) with ROS were again only evident in African men. ROS is positively associated with SBP and PP in African men, suggesting that increased ROS levels may contribute to hypertension in this population group.

The involvement of metallothioneins in mitochondrial function and disease.

Mitochondrial oxidative phosphorylation deficiency is accompanied by various down-stream, adaptive responses which play a key role in the varied phenotypes observed when mitochondrial dysfunction occurs. These responses are often accompanied by the induction of genes involved in defense mechanisms against oxidative stress. Among these responses, metallothioneins (MTs) has been identified to be responsive to mitochondrial dysfunction. MTs, which are expressed in four different isoforms, are small, cysteine rich, metal binding proteins that have been associated with a protective effect in cells under numerous diseased and stressed states. Their diverse functionality and protective roles can be ascribed to their three basic abilities or primary functions which are metal homeostasis, heavy metal detoxification and free radical scavenging. The involvement of MTs with numerous cellular processes, organelles and cells has received much attention while notice of their involvement with the function of mitochondria has been lacking. It is believed that MTs promote the survival of mitochondrial dysfunctional cells by acting as highly efficient reducing elements against the damaging properties of reactive oxygen species (ROS) and by limiting apoptosis. In addition to their role in mitochondrial disease, convincing evidence exist, albeit with conflicting results, of its involvement in some key functions of the mitochondrion, including redox modulation, metal homeostasis and enzyme and transcription factor regulation.

DNA damage and repair detected by the comet assay in lymphocytes of african petrol attendants: a pilot study.

Petrol attendants are exposed to petrol volatile organic compounds (VOCs) which may have genotoxic and carcinogenic effects. The single-cell gel electrophoresis assay (comet assay) is a method highly sensitive to DNA damage induced by environmental and occupational exposure to carcinogenic and mutagenic agents. The aim of this study was to evaluate the level of exposure of petrol attendants to petrol VOCs and also to determine their effect on DNA damage and repair in lymphocytes of African petrol attendants. The exposed group consisted of 20 subjects, randomly selected from three petrol stations. A control group of 20 unexposed subjects was also chosen and matched for age and smoking habits with the exposed group. Sorbent tubes were used to assess personal exposure of petrol attendants. The comet assay was used to investigate the basal DNA damage and repair capacity in isolated lymphocytes of petrol attendants and unexposed subjects. Blood samples were taken from the petrol attendants at the end of their 8-h working shift and also from the unexposed subjects. The petrol attendants were found to be exposed to levels of petrol VOCs lower than the South African occupational exposure limit for constituent chemicals. A significant relationship was found between the volume of petrol sold during the shift and the average concentrations of benzene, toluene and the total VOCs measured. However, relative humidity had a negative correlation with the average concentrations of benzene, toluene, xylene and the total VOCs. Significantly higher basal DNA damage was observed with the exposed group compared to the unexposed group. The period of exposure influenced the level of DNA damage and the calculated repair capacity. Smoking and age had a significant influence on the level of DNA damage. DNA repair capacity was delayed in smokers of both exposed and unexposed group.

Human mitochondrial complex I deficiency: investigating transcriptional responses by microarray.

NADH:ubiquinone oxidoreductase (complex I) deficiency is one of the most frequently encountered defects of the mitochondrial energy generating system. A deficiency of this enzyme complex leads to a wide variety in clinical disease expression. The cell biological consequences of such mutations, however, are poorly understood. We investigated transcriptional responses in fibroblast cell lines harboring mutations in the five different nuclear DNA encoded subunits using a mitochondria-targeting microarray. Expression profiles of cell lines cultured under conditions that favor glycolytic metabolism were compared to profiles when cultured under conditions favoring oxidative metabolism. Approximately 60 genes displayed differential expression under these conditions in either all mutated cell lines or selected cell lines only. A marked induction of metallothioneins as well as ATP1G1 transcripts was detected in all patient cell lines. Transcriptional responses such as the induction of heat shock protein transcripts, decreased PDK1,BNIP3 and mitochondrial genome encoding gene transcripts occurred in selected patient cell lines. The observed transcript profile points to a common, putative defensive, response relating to oxidative stress. Although further investigations of other human OXPHOS system diseases is warranted, these results clearly underline that functional genomics holds for the study of inherited metabolic disease.

A kinetic study into the hydrolysis of the ochratoxins and analogues by carboxypeptidase A.

The hydrolyses of the ochratoxins and analogues by carboxypeptidase A were assessed. This was done by measuring the amount of phenylalanine formed with liquid chromatography coupled to tandem electrospray mass spectrometry. The kinetic data of ochratoxin A, ochratoxin B, and the synthetic bromo-ochratoxin B were compared to the values of a number of synthesized structure analogues, namely, ochratoxin A methyl ester, ochratoxin B methyl ester, N-(2-hydroxybenzoyl)phenylalanine, N-(5-chloro-2-hydroxybenzoyl)phenylalanine, N-(5-bromo-2-hydroxybenzoyl)phenylalanine, and N-(5-fluoro-2-hydroxybenzoyl)phenylalanine. The halogen-containing analogues had lower turnovers than their des-halo analogues. There are no substantial differences in the kinetic data between the different halogen-containing analogues.

The utilization of alanine, glutamic acid, and serine as amino acid substrates for glycine N-acyltransferase.

The conjugation of benzoyl-CoA with the aliphatic and acidic amino acids by glycine N-acyltransferase, as well as the amides of the latter group, was investigated. Bovine and human liver benzoyl-amino acid conjugation were investigated using electrospray ionization tandem mass spectrometry (ESI-MS-MS). Bovine glycine N-acyltransferase catalyzed conjugation of benzoyl-CoA with Gly (Km(Gly) = 6.2 mM), Asn (Km(Asn) = 129 mM), Gln (Km(Gln) = 353 mM), Ala (Km(Ala) = 1573 mM), Glu (Km(Glu) = 1148 mM) as well as Ser in a sequential mechanism. In the case of the human form, conjugation with Gly (Km(Gly) = 6.4 mM), Ala (Km(Ala) = 997 mM), and Glu was detected. The presence of these alternative conjugates did not inhibit bovine glycine N-acyltransferase activity significantly. Considering the relatively low levels at which these conjugates are formed, it is unlikely that they will have a significant contribution to acyl-amino acid conjugation under normal conditions in vivo. However, their cumulative contribution to acyl-amino acid conjugation under metabolic disease states may prove to have a useful contribution to detoxification of elevated acyl-CoAs.

Carnitine palmitoyltransferase I activity monitoring in fibroblasts and leukocytes using electrospray ionization mass spectrometry.

Carnitine palmitoyltransferase I (CPT I) is one of the enzymes associated with normal mitochondrial membrane transport of certain metabolites. The importance of the enzyme in normal energy production is well illustrated during fasting conditions when a large flux of long-chain fatty acids must be transported over the mitochondrial membrane to undergo beta-oxidation. Up to now CPT I activity has been assayed in various tissues, including liver, leukocytes, platelets, and fibroblasts by the use of an isotope exchange forward assay which measures the rate of palmitoyl-l-[methyl-3H]carnitine formation from palmitoyl-CoA and l-[methyl-3H]carnitine. We have developed an electrospray ionization mass spectrometric method for detecting palmitoylcarnitine formation from palmitoyl-CoA and carnitine, thus avoiding the use of radiolabeled isotopes. In this assay, time-dependent conversion of free carnitine by CPT I to palmitoylcarnitine is measured quantitatively, relative to isotopically labelled palmitoylcarnitine, by parent ion monitoring of fragment ion m/z 85. The specific activity of CPT I in fibroblasts and leukocytes compared well with the activity determined with the isotope exchange method, however, the combination of high sensitivity and selectivity of tandem mass spectrometry along with the environment-friendly nature of the electrospray method makes it an ideal technique to measure CPT I activity.

Contribution of regions 3' and 5' to the hIL-5 gene on the expression of rhIL-5 in CHO-cells.

The effect of the presence of the regions 5' and 3' of the hIL-5 gene on the expression of recombinant hIL-5 in CHO-cells was investigated. The 3.2 kb hIL-5 gene-fragment was cloned and four different dexamethasone-inducible rhIL-5 mammalian expression-vectors were constructed, each containing a different part of the gene-fragment. Results indicated that deletion of the region 5' to the gene increases production three-fold whereas a four-fold decrease in production was observed with deletion of the region 3' to the gene. Deletion of both regions increased rhIL-5 production by only one and a half-fold. These results indicate that there are elements in a 928 bp region 3' to the gene, including the 3'-NTR, that have an enhancing or stabilizing effect on expression of hIL-5 in CHO-cells.