Head and neck squamous-cell cancer and its association with polymorphic enzymes of xenobiotic metabolism and repair

Tobacco smoking, alcohol drinking, and occupational exposures to polycyclic aromatic hydrocarbons are the major proven risk factors for human head and neck squamous-cell cancer (HNSCC). Major research focus on gene-environment interactions concerning HNSCC has been on genes encoding enzymes of metabolism for tobacco smoke constituents and repair enzymes. To investigate the role of genetically determined individual predispositions in enzymes of xenobiotic metabolism and in repair enzymes under the exogenous risk factor tobacco smoke in the carcinogenesis of HNSCC, we conducted a case-control study on 312 cases and 300 noncancer controls. We focused on the impact of 22 sequence variations in CYP1A1, CYP1B1, CYP2E1, ERCC2/XPD, GSTM1, GSTP1, GSTT1, NAT2, NQO1, and XRCC1. To assess relevant main and interactive effects of polymorphic genes on the susceptibility to HNSCC we used statistical models such as logic regression and a Bayesian version of logic regression. In subgroup analysis of nonsmokers, main effects in ERCC2 (Lys751Gln) C/C genotype and combined ERCC2 (Arg156Arg) C/A and A/A genotypes were predominant. When stratifying for smokers, the data revealed main effects on combined CYP1B1 (Leu432Val) C/G and G/G genotypes, followed by CYP1B1 (Leu432Val) G/G genotype and CYP2E1 (-70G>T) G/T genotype. When fitting logistic regression models including relevant main effects and interactions in smokers, we found relevant associations of CYP1B1 (Leu432Val) C/G genotype and CYP2E1 (-70G>T) G/T genotype (OR, 10.84; 95% CI, 1.64-71.53) as well as CYP1B1 (Leu432Val) G/G genotype and GSTM1 null/null genotype (OR, 11.79; 95% CI, 2.18-63.77) with HNSCC. The findings underline the relevance of genotypes of polymorphic CYP1B1 combined with exposures to tobacco smoke.

Influence of polymorphisms of the human glutathione transferases and cytochrome P450 2E1 enzyme on the metabolism and toxicity of ethylene oxide and acrylonitrile

A cohort of 59 persons with industrial handling of low levels of acrylonitrile is being studied as part of a medical surveillance programme. Previously, an extended haemoglobin adduct monitoring (N-(cyanoethyl)valine and N-(hydroxyethyl)-valine) was performed regarding the glutathione transferases hGSTM1 and hGSTT1 polymorphisms but no influence of hGSTM1 or hGSTT1 polymorphisms on specific adduct levels was found. A compilation of case reports of human accidental poisonings had pointed to significant individual differences in human acrylonitrile metabolism and toxicity. Therefore, a re-evaluation of the industrial cohort included known polymorphisms of the glutathione transferases hGSTM3 and hGSTP1 as well as of the cytochrome P450 CYP2E1. A detailed statistical analysis revealed that exposed carriers of the allelic variants of hGSTP1, hGSTP1*B/hGSTP1*C, characterized by a single nucleotide polymorphism at nucleotide 313 which results in a change from Ile to Val at codon 104, had higher levels of the acrylonitrile-specific haemoglobin adduct N-(cyanoethyl)valine compared to the carriers of the codon 113 alleles hGSTP1*A and hGSTP1*D. The single nucleotide polymorphism at codon 113 of hGSTP1 (hGSTP1*A/hGSTP1*B versus hGSTP1*C/hGSTP1*D) did not show an effect, and also no influence was seen on specific haemoglobin adduct levels of the polymorphisms of hGSTM3 or CYP2E1. The data, therefore, point to a possible influence of a human enzyme polymorphism of the GSTP1 gene at codon 104 on the detoxication of acrylonitrile which calls for experimental toxicological investigation. The study also confirmed the impact of GSTT1 polymorphism on background N-(hydroxyethyl)-valine adduct levels in haemoglobin which are caused by endogenous ethylene oxide.

Species differences in acrylonitrile metabolism and toxicity between experimental animals and humans based on observations in human accidental poisonings

The high acute toxicity of acrylonitrile may be a result of its intrinsic biological reactivity or of its metabolite cyanide. Intravenous N-acetylcysteine has been recommended for treatment of accidental intoxications in acrylonitrile workers, but such recommendations vary internationally. Acrylonitrile is metabolized in humans and experimental animals via two competing pathways; the glutathione-dependent pathway is considered to represent an avenue of detoxication whilst the oxidative pathway leads to a genotoxic epoxide, cyanoethylene oxide, and to elimination of cyanide. Cases of acute acrylonitrile overexposure or intoxication have occurred within persons having industrial contact with acrylonitrile; the route of exposure was by inhalation and/or by skin contact. The combined observations lead to the conclusion of a much higher impact of the oxidative metabolism of acrylonitrile in humans than in rodents. This is confirmed by differences in the clinical picture of acute life-threatening intoxications in both species, as well as by differential efficacies of antidotes. A combination of N-acetylcysteine with sodium thiosulfate seems an appropriate measure for antidote therapy of acute acrylonitrile intoxications. Clinical observations also highlight the practical importance of human individual susceptibility differences. Furthermore, differential adduct monitoring, assessing protein adducts with different rates of decay, enables the development of more elaborated biological monitoring strategies for the surveillance of workers with potential acrylonitrile contact.

Possible impact of human CYP2E1 polymorphisms on the metabolism of acrylonitrile

Case reports of human accidental poisonings point to significant individual differences in human acrylonitrile metabolism and toxicity. A cohort of 59 persons with industrial handling of low levels of acrylonitrile has repetitively been studied from 1994 through 1999 as part of a medical surveillance programme. The analyses included adduct determinations of N-terminal N-(cyanoethyl)valine in haemoglobin and genotypings of the following cytochrome P-450 2E1 (CYP2E1) polymorphisms: G-1259C and C-1019T (two subjects heterozygous), A-316G (three subjects heterozygous), T-297A (15 subjects heterozygous), G-35T (eight subjects heterozygous), G4804A (two subjects heterozygous), T7668A (six subjects heterozygous). N-(Cyanoethyl)valine adduct levels were, if any, only slightly influenced by smoking and mainly determined by the external acrylonitrile exposures. The individual means and medians of N-(cyanoethyl)valine levels over the entire observation period were compared with the CYP2E1 variants (Wilcoxon rank sum test). No influences of the investigated CYP2E1 polymorphisms on the N-(cyanoethyl)valine levels appeared at the 5% level. However, there was a trend, at a level of P≃0.1, pointing to higher acrylonitrile-specific adduct levels in persons with the A-316G mutation. Higher adduct levels would be compatible with a slower CYP2E1-mediated metabolism of acrylonitrile and with lower extents of toxification to cyanide.

Weight-related differences in glucose metabolism and free fatty acid production in two South African population groups

OBJECTIVE The effects of free fatty acids (FFA), leptin, tumour necrosis factor (TNF) alpha and body fat distribution on in vivo oxidation of a glucose load were studied in two South African ethnic groups. DESIGN AND MEASUREMENTS Anthropometric and various metabolic indices were measured at fasting and during a 7h oral glucose tolerance test (OGTT). Body composition was measured using bioelectrical impedance analysis and subcutaneous and visceral fat mass was assessed using a five- and two-level CT-scan respectively. Glucose oxidation was evaluated by measuring the ratio of (13)CO(2) to (12)CO(2) in breath following ingestion of 1-(13)C-labelled glucose. SUBJECTS Ten lean black women (LBW), ten obese black women (OBW), nine lean white women (LWW) and nine obese white women (OWW) were investigated after an overnight fast. RESULTS Visceral fat levels were significantly higher (P < 0.01) in obese white than black women, despite similar body mass indexes (BMIs). There were no ethnic differences in glucose oxidation however; in the lean subjects of both ethnic groups the area under the curve (AUC) was higher than in obese subjects (P < 0.05 for both) and was found to correlate negatively with weight (r = -0.69, P < 0.01) after correcting for age. Basal TNF alpha concentrations were similar in all groups. Percentage suppression of FFAs at 30 min of the OCTT was 24 +/- 12% in OWW and - 38 +/- 23% (P < 0.05) in OBW, ie the 30 min FFA level was higher than the fasting level in the latter group. AUC for FFAs during the late postprandial period (120 - 420 min) was significantly higher in OWW than OBW (P < 0.01) and LWW (P < 0.01) and correlated positively with visceral fat mass independent of age (r = 0.78, P < 0.05) in the OWW only. Leptin levels were higher (P < 0.01) both at fasting and during the course of the OCTT in obese women from both ethnic groups compared to the lean women. CONCLUSIONS Glucose oxidation is reduced in obese subjects of both ethnic groups; inter- and intra-ethnic differences were observed in visceral fat mass and FFA production and it is possible that such differences may play a role in the differing prevalences of obesity-related disorders that have been reported in these two populations.

Ethnic differences in lipid metabolism in two groups of obese South African women

There is a higher prevalence of ischemic heart disease (IHD) in South African white than black women. The objective of this study was to determine biochemical explanations for this prevalence. The study group contained 15 obese black women (OBW) and 14 obese white women (OWW), ah premenopausal, who were examined after an overnight fast. Anthropometric measurements and blood concentrations of glucose, non-esterified fatty acids (NEFAs), catecholamines, plasminogen activator inhibitor-1, C-peptide, proinsulin, lipograms, cortisol, growth hormone, and post-heparin Lipoprotein Lipase activity were measured during an oral glucose tolerance test (OGTT), Body composition was measured using bioelectrical impedance analysis, and subcutaneous and visceral fat mass were assessed with CT-scans. Visceral fat area was higher in OWW (139.7 +/- 10.7 cm(2)) than in OBW (72.3 +/- 3.9 cm(2)) (P < 0.01), as were fasting and 3 h triglyceride concentrations (P < 0.05 for all). OWW also had higher NEFA levels than OBW at 3 and 4 h compared, with OBW (P < 0.05 for both). Fasting cortisol (266 +/- 24 vs. 197 +/- 19 nmol/l; P < 0.05) was higher in OWW than in OBW. These data demonstrate that OWW have higher visceral fat mass than OBW, which may lead to a more atherogenic fasting and postprandial Lipid profile. The higher cortisol levels of the OWW may promote visceral fat deposition. - Punyadeera, C., M-T. van der Merwe, N.J. Crowther, M. Toman, C. P. Schlaphoff, and I. P. Gray. Ethnic differences in lipid metabolism in two groups of obese South African women.

AICAR administration affects glucose metabolism by upregulating the novel glucose transporter, GLUT8, in equine skeletal muscle

Equine metabolic syndrome is characterized by obesity and insulin resistance (IR). Currently, there is no effective pharmacological treatment for this insidious disease. Glucose uptake is mediated by a family of glucose transporters (GLUT), and is regulated by insulin-dependent and -independent pathways, including 5-AMP-activated protein kinase (AMPK). Importantly, the activation of AMPK, by 5-aminoimidazole- 4-carboxamide-1-D-ribofuranoside (AICAR) stimulates glucose uptake in both healthy and diabetic humans. However, whether AICAR promotes glucose uptake in horses has not been established. It is hypothesized that AICAR administration would enhance glucose transport in equine skeletal muscle through AMPK activation. In this study, the effect of an intravenous AICAR infusion on blood glucose and insulin concentrations, as well as on GLUT expression and AMPK activation in equine skeletal muscle (quantified by Western blotting) was examined. Upon administration, plasma AICAR rapidly reached peak concentration. Treatment with AICAR resulted in a decrease (P < 0.05) in blood glucose and an increase (P < 0.05) in insulin concentration without a change in lactate concentration. The ratio of phosphorylated to total AMPK was increased (P < 0.05) in skeletal muscle. While GLUT4 and GLUT1 protein expression remained unchanged, GLUT8 was increased (P < 0.05) following AICAR treatment. Up-regulation of GLUT8 protein expression by AICAR suggests that this novel GLUT isoform plays an important role in equine muscle glucose transport. In addition, the data suggest that AMPK activation enhances pancreatic insulin secretion. Collectively, the findings suggest that AICAR acutely promotes muscle glucose uptake in healthy horses and thus its therapeutic potential for managing IR requires investigation.

Light history-dependent respiration explains the hysteresis in the daily ecosystem metabolism of seagrass

Oxygen flux between aquatic ecosystems and the water column is a measure of ecosystem metabolism. However, the oxygen flux varies during the day in a “hysteretic” pattern: there is higher net oxygen production at a given irradiance in the morning than in the afternoon. In this study, we investigated the mechanism responsible for the hysteresis in oxygen flux by measuring the daily pattern of oxygen flux, light, and temperature in a seagrass ecosystem (Zostera muelleri in Swansea Shoals, Australia) at three depths. We hypothesised that the oxygen flux pattern could be due to diel variations in either gross primary production or respiration in response to light history or temperature. Hysteresis in oxygen flux was clearly observed at all three depths. We compared this data to mathematical models, and found that the modification of ecosystem respiration by light history is the best explanation for the hysteresis in oxygen flux. Light history-dependent respiration might be due to diel variations in seagrass respiration or the dependence of bacterial production on dissolved organic carbon exudates. Our results indicate that the daily variation in respiration rate may be as important as the daily changes of photosynthetic characteristics in determining the metabolic status of aquatic ecosystems.

Cuff tear arthropathy: Evidence of functional variation in pyrophosphate metabolism genes

We investigated the role of two genes, ANKH and TNAP, in patients with cuff tear arthropathy. These genes encode proteins which regulate the extracellular concentration of inorganic pyrophosphate, fluctuations of which can lead to calcium crystal formation. Variants were detected by direct sequencing of DNA and their frequencies compared with healthy controls. The effect of variants on protein function was further studied by in vitro approaches. Variant genotypes were observed more frequently in the cases when compared with controls in ANKH (45% and 20%) and TNAP (32% and 9%). Variants in ANKH altered inorganic pyrophosphate (PPi) concentrations in transfected human chondrocytes. There was a higher mean serum concentration of TNAP detected in female patients compared with normal ranges. Cuff tear arthropathy is associated with variants in ANKH and TNAP that alter extracellular inorganic pyrophosphate concentrations causing calcium crystal deposition. This supports a theory that genetic variants predispose patients to primary crystal deposition which when combined with a massive rotator cuff tear leads to the development of arthritis.

UVB irradiation alters the activities and kinetic properties of the enzymes of energy metabolism in rat lens during aging

Ultraviolet (UV) radiation is one of the major risk factors of cataract (loss of eye-lens transparency). The influence of UVB radiation (300 nm, 100 mu W cm(-2)) on the activity and apparent kinetic constants (K-m and V-max) of rat lens hexokinase (HK;EC2.7.1.1), phosphofructokinase (PFK;EC2.7.1.11), isocitrate dehydrogenase (ICDH;EC1.1.1.41) and malate dehydrogenase (MDH;EC1.1.1.37) of energy metabolism has been investigated by irradiating the lens homogenate of three-and 12-month-old rats. In the three-month-old group specific activities of HK and PFK are reduced by 56 and 43 %, respectively, and there is no change in ICDH and MDH activities after a 24 h exposure. On the other hand, in the 12-month-old group the decreases are 72, 71, 24 and 16 % for HK, PFK. ICDH and MDH, respectively. UVB irradiation increases the apparent K-m of HK and PFK (in both age groups), whereas the K-m of ICDH and MDH is not altered. While the decrease in V-max of these enzymes due to UVB exposure is only marginal in three-month-old rats, it is more pronounced (significant) in 12-month-old rats. A similar decrease in enzyme activities of HK and PFK is also observe upon UVB exposure of the intact rat lens. The photoinduced changes in energy metabolism may in turn have a bearing on lens transparency, particularly at an older age.

Liquid Chromatography-Mass Spectrometry in Studies of Drug Metabolism and Permeability

Poor pharmacokinetics is one of the reasons for the withdrawal of drug candidates from clinical trials. There is an urgent need for investigating in vitro ADME (absorption, distribution, metabolism and excretion) properties and recognising unsuitable drug candidates as early as possible in the drug development process. Current throughput of in vitro ADME profiling is insufficient because effective new synthesis techniques, such as drug design in silico and combinatorial synthesis, have vastly increased the number of drug candidates. Assay technologies for larger sets of compounds than are currently feasible are critically needed. The first part of this work focused on the evaluation of cocktail strategy in studies of drug permeability and metabolic stability. N-in-one liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods were developed and validated for the multiple component analysis of samples in cocktail experiments. Together, cocktail dosing and LC/MS/MS were found to form an effective tool for increasing throughput. First, cocktail dosing, i.e. the use of a mixture of many test compounds, was applied in permeability experiments with Caco-2 cell culture, which is a widely used in vitro model for small intestinal absorption. A cocktail of 7-10 reference compounds was successfully evaluated for standardization and routine testing of the performance of Caco-2 cell cultures. Secondly, cocktail strategy was used in metabolic stability studies of drugs with UGT isoenzymes, which are one of the most important phase II drug metabolizing enzymes. The study confirmed that the determination of intrinsic clearance (Clint) as a cocktail of seven substrates is possible. The LC/MS/MS methods that were developed were fast and reliable for the quantitative analysis of a heterogenous set of drugs from Caco-2 permeability experiments and the set of glucuronides from in vitro stability experiments. The performance of a new ionization technique, atmospheric pressure photoionization (APPI), was evaluated through comparison with electrospray ionization (ESI), where both techniques were used for the analysis of Caco-2 samples. Like ESI, also APPI proved to be a reliable technique for the analysis of Caco-2 samples and even more flexible than ESI because of the wider dynamic linear range. The second part of the experimental study focused on metabolite profiling. Different mass spectrometric instruments and commercially available software tools were investigated for profiling metabolites in urine and hepatocyte samples. All the instruments tested (triple quadrupole, quadrupole time-of-flight, ion trap) exhibited some good and some bad features in searching for and identifying of expected and non-expected metabolites. Although, current profiling software is helpful, it is still insufficient. Thus a time-consuming largely manual approach is still required for metabolite profiling from complex biological matrices.

Expression Profiling of Nucleotide Metabolism-Related Genes in Human Breast Cancer Cells After Treatment with 5-Fluorouracil

5-Fluorouracil (5-FU) is one of the most widely used drugs for treatment of cancers, including breast cancer that exhibits its anticancer activity by inhibiting DNA synthesis and also incorporated into DNA and RNA. The objective of this investigation was to find out the total nucleotide metabolism genes regulated by 5-FU in breast cancer cell line. The breast cancer cell line MCF-7 was treated with the drug 5-FU. To analyze the expression of genes, we have conducted the experiment using 1.7k and 19k human microarray slide and confirmed the expression of genes by semiquantitative reverse transcription-polymerase chain reaction. The expression of 44 genes involved in the nucleotide metabolism pathway was quantified. Of these 44 genes analyzed, transcription of 6 genes were upregulated and 9 genes were downregulated. Earlier studies revealed that the transcription of genes for key enzymes like thymidylate synthase, thymidinekinase, and dihydropyrimidine dehydrogenase are regulated by 5-FU. This study identified some novel genes like thioredoxin reductase, ectonucleotide triphosphate dephosphorylase, and CTP synthase are regulated by 5-FU. The data also reveal large-scale perturbation in transcription of genes not involved directly in the known mechanism of action of 5-FU.

Metabolism of 1,8-Cineole in Rat: Its Effects on Liver and Lung Microsomal Cytochrome P-450 Systems

The monoterpene cyclic ether, cineole (l,8-cineole, I) also known as eucalyptol, is a component of many essential oils and is widely distributed in nature. It is extensively used in pharmaceutical preparations for external application and also as a nasal spray. It was reported earlier that cineole when administered to sheep may be largely oxidized in the system (Scheline 1978). However the mode of metabolism of cineole is not known. Hence the present study was undertaken to investigate the metabolic fate of this ubiquitous terpenoid following its administration to rats by gastric intubation.

Partial purification and properties of a transamidinase from Lathyrus sativus seedlings. Involvement in homoarginine metabolism and amine interconversions

A transamidinase was purified 463-fold from Lathyrus sativus seedlings by affinity chromatography on homoarginine--Sepharose. The enzyme exhibited a wide substrate specificity, and catalysed the reversible transfer of the amidino groups from donors such as arginine, homoarginine and canavanine to acceptors such as lysine, putrescine, agmatine, cadaverine and hydroxylamine. The enzyme could not be detected in the seeds, and attained the highest specific activity in the embryo axis on day 10 after seed germination. Its thiol nature was established by strong inhibition by several thiol blockers and thiol compounds in the presence of ferricyanide. In the absence of an exogenous acceptor, it exhibited weak hydrolytic activity towards arginine. It had apparent mol.wt. 210000, and exhibited Michaelis--Menten kinetics with Km 3.0 mM for arginine. Ornithine competitively inhibited the enzyme, with Ki 1.0 mM in the arginine--hydroxylamine amidino-transfer reaction. Conversion experiments with labelled compounds suggest that the enzyme is involved in homoarginine catabolism during the development of plant embryo to give rise to important amino acids and amine metabolites. Presumptive evidence is also provided for its involvement in the biosynthesis of the guanidino amino acid during seed development. The natural occurrence of arcain in L. sativus and mediation of its synthesis in vitro from agmatine by the transamidinase are demonstrated.