Enzymes involved in the bioactivation of 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone in patas monkey lung and liver microsomes

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco-specific carcinogen in animals. Our previous studies indicated that there are differences between rodents and humans for the enzymes involved in the activation of NNK. To determine if the patas monkey is a better animal model for the activation of NNK in humans, we investigated the metabolism of NNK in patas monkey lung and liver microsomes and characterized the enzymes involved in the activation. In lung microsomes, the formation of 4-oxo-1-(3-pyridyl)-1-butanone (keto aldehyde), 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone (NNK- N-oxide), 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol), and 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was observed, displaying apparent Km values of 10.3, 5.4, 4.9, and 902 microM, respectively. NNK metabolism in liver microsomes resulted in the formation of keto aldehyde, keto alcohol, and NNAL, displaying apparent Km values of 8.1, 8.2, and 474 microM, respectively. The low Km values for NNK oxidation in the patas monkey lung and liver microsomes are different from those in human lung and liver microsomes showing Km values of 400-653 microM, although loss of low Km forms from human tissue as a result of disease, surgery or anesthesia cannot be ruled out. Carbon monoxide (90%) significantly inhibited NNK metabolism in the patas monkey lung and liver microsomes by 38-66% and 82-91%, respectively. Nordihydroguaiaretic acid (a lipoxygenase inhibitor) and aspirin (a cyclooxygenase inhibitor) decreased the rate of formation of keto aldehyde and keto alcohol by 10-20 % in the monkey lung microsomes. Alpha-Napthoflavone and coumarin markedly decreased the oxidation of NNK in monkey lung and liver microsomes, suggesting the involvement of P450s 1A and 2A6. An antibody against human P450 2A6 decreased the oxidation of NNK by 12-16% and 22-24% in the patas monkey lung and liver microsomes, respectively. These results are comparable to that obtained with human lung and liver microsomes. Coumarin hydroxylation was observed in the patas monkey lung and liver microsomes at a rate of 16 and 4000 pmol/min/mg protein, respectively, which was 5-fold higher than human lung and liver microsomes, respectively. Immunoblot analysis demonstrated that the P450 2A level in the individual patas monkey liver microsomal sample was 6-fold greater than in an individual human liver microsomal sample. Phenethyl isothiocyanate, an inhibitor of NNK activation in rodents and humans, decreased NNK oxidation in the monkey lung and liver microsomes displaying inhibitor concentration resulting in 50% inhibition of the activity (IC50) values of 0.28-0.8 microM and 4.2-6.8 microM, respectively. The results demonstrate the similarities and differences between species in the metabolic activation of NNK. The patas monkey microsomes appear to more closely resemble human microsomes than mouse or rat enzymes and may better reflect the activation of NNK in humans.      

Dichloroacetic acid reduces Ha-ras codon 61 mutations in liver tumors from female B6C3F1 mice

Dichloroacetic acid (DCA), a disinfection by-product of chlorination found in drinking water, is a hepatocarcinogenic in the B6C3F1 mouse. Previous studies have shown that DCA does not significantly alter the incidence of Ha-ras codon 61 mutations in male mouse liver carcinomas from that observed in spontaneous tumors (approximately 50% have Ha-ras mutations) but it alters the proportions of mutations that occur in Ha- ras codon 61. Twenty-two tumors were produced in female B6C3F1 mice after treatment with 3.5 g DCA per liter of drinking water over a period of 104 weeks. To detect potential Ha-ras mutations in the liver tumor tissue of female B6C3F1 mice, genomic DNA was isolated from tumors that had been frozen. The polymerase chain reaction (PCR) and single-stranded conformational polymorphism (SSCP) was used to screen tumor DNA for mutations in Ha-ras exon 2. In DNA from liver tumors in female B6C3F1 mice induced by DCA-treatment we found only one mutation in exon 2 among the 22 tumors analyzed (4.5%). Direct-sequencing of exon 2 revealed a CAA to CTA transversion in Ha-ras codon 61. The result of this study indicates that tumor formation in DCA-treated female B6C3F1 mice is, therefore, not associated with a mutationally activated Ha-ras codon 61. This result differs from previous results obtained in male B6C3F1 mice.      

Highly-efficient electrotransformation of the yeast Hansenula polymorpha

A highly-efficient method for transformation of the methylotrophic yeast Hansenula polymorpha has been developed. Routinely, transformation frequencies of up to 1.7×10⁶/g plasmid DNA were obtained by applying an electric pulse of the exponential decay type of 7.5 kV/cm to a highly-concentrated cell mixture during 5 ms. Efficient transformation was dependent on: (1) pretreatment of the cells with the reducing agent dithiotreitol, (2) the use of sucrose as an osmotic stabilizer in an ionic electroporation buffer, and (3) the use of cells grown to the mid-logarithmic phase. Important parameters for optimizing the transformation frequencies were field strength, pulse duration, and cell concentration during the electric pulse. In contrast to electrotransformation protocols described for Saccharomyces cerevisiae and Candida maltosa, transformation frequencies (transformants per g DNA) for H. polymorpha remained high when large amounts (up to 10g) of plasmid DNA were added. This feature renders this procedure pre-eminently advantageous for gene cloning experiments when high numbers of transformants are needed.     

An Australian childhood obesity summit: the role of data and evidence in 'public' policy making

Background

Overweight and obesity in Australia has risen at an alarming rate over the last 20 years as in other industrialised countries around the world, yet the policy response, locally and globally, has been limited. Using a childhood obesity summit held in Australia in 2002 as a case study, this paper examines how evidence was used in setting the agenda, influencing the Summit debate and shaping the policy responses which emerged. The study used multiple methods of data collection including documentary analysis, key informant interviews, a focus group discussion and media analysis. The resulting data were content analysed to examine the types of evidence used in the Summit and how the state of the evidence base contributed to policy-making.

Results

Empirical research evidence concerning the magnitude of the problem was widely reported and largely uncontested in the media and in the Summit debates. In contrast, the evidence base for action was mostly opinion and ideas as empirical data was lacking. Opinions and ideas were generally found to be an acceptable basis for agreeing policy action coupled with thorough evaluation. However, the analysis revealed that the evidence was fiercely contested around food advertising to children and action agreed was therefore limited.

Conclusion

The Summit demonstrated that policy action will move forward in the absence of strong research evidence. Where powerful and competing groups contest possible policy options, however, the evidence base required for action needs to be substantial. As with tobacco control, obesity control efforts are likely to face ongoing challenges around the nature of the evidence and interventions proposed to tackle the problem. Overcoming the challenges in controlling obesity will be more likely if researchers and public health advocates enhance their understanding of the policy process, including the role different types of evidence can play in influencing public debate and policy decisions, the interests and tactics of the different stakeholders involved and the part that can be played by time-limited yet high profile events such as Summits.

     

Human Exposure to Styrene Vapor

Human volunteers were exposed to styrene vapor at approximately 50, 100, 200, and 375 ppm for periods of one to seven hours. Only at 375 ppm did the subjects experience significant subjective symptoms and show objective signs of transient neurological impairment. A small portion of the absorbed styrene was rapidly and exponentially excreted from the lungs in the postexposure period. A measurement of the amount of styrene present in the breath in the early postexposure period provided a means for making an estimation of magnitude of exposure. The determination of urinary hippuric acid proved to be insensitive as an indicator of exposure to this compound at these vapor concentrations.     

Experience with autologous pericardial patch closure of ventricular septal defect

Background Conventionally, Ventricular Septal Defects (VSDs) are repaired with synthetic patch—Dacron (polyethylene terephthalate) or Goretex (expanded polytetrafluoroethylene). Recently, we began using glutaraldehyde—treated autologous pericardial patch to repair VSDs. We review our experience. Material and Method Between July to November 2005, 60 children had their VSDs repaired with glutaraldehyde—treated autologous pericardium. There were 40 males and 20 females, aged between 5 months and 12 years with a median age of 1 year. The diagnosis was isolated VSD in 37 patients, multiple VSD in 3; Tetralogy of Fallot (TOF) in 15 and Double Outlet Right Ventricle (DORV) in 5 patients. The chest was opened by a median sternotomy incision. After establishing cardiopulmonary bypass, a strip of pericardium was harvested from the patient and fixed in 0.6% glutaraldehyde (Polyscientific, Bayshore, NY) for about 20 minutes. It was then washed out with 0.9% saline solution. The defect was repaired with 4/0 or 5/0 prolene suture using a continuous suture technique. Results There was no hospital mortality. Postoperative echocardiogram revealed trivial shunts in 10 patients. Follow up was for 3 to 6 months (mean 2 months). No patient required reoperation for residual VSD. Conclusion Glutaraldehyde—treated autologous pericardium is an excellent material for surgical patch clousre of VSD. It is easily available and does not require sterilization. Further follow-up is required to assess its long term efficacy.     

Supraorbital neuralgia: a clinical study

A series of 18 patients suffering from supraorbital neuralgia have been studied through a seven year period. Appropriate investigations ruled out other headaches. There was a female (67%) preponderance. Mean age at onset was 51.6 years. The mean headache duration was 5.9 years. Five patients had a history of ipsilateral forehead trauma. The main areas of pain were the forehead and orbit. The pain was dull with short sharp or burning exacerbations. The temporal pattern was either remitting (n = 7) or chronic continuous (n = 11). Autonomic accompaniments were generally lacking. Neurological assessment was normal in all but 4 patients who were found to have signs/symptoms of sensory dysfunction in the forehead of the symptomatic side. Trials of different drugs, including migraine and anti-neuralgic drugs, only provided slight relief. Anaesthetic nerve blocks of the supraorbital nerve provided an absolute but transitory relief of pain. Although aetiology and pathogenesis of supraorbital neuralgia is largely unknown, entrapment of the supraorbital nerve at its outlet and successful decompressive surgery have been previously reported. This and other pathogenic hypotheses are discussed.     

A systematic review with procedural assessments and meta-analysis of Low Level Laser Therapy in lateral elbow tendinopathy (tennis elbow)

Background  

Recent reviews have indicated that low level level laser therapy (LLLT) is ineffective in lateral elbow tendinopathy (LET) without assessing validity of treatment procedures and doses or the influence of prior steroid injections.