Modified nicotine metabolism in transgenic tobacco plants expressing the human cytochrome P450 2A6 cDNA

In this study, the human cytochrome P450 (CYP) 2A6 was used in order to modify the alkaloid production of tobacco plants. The cDNA for human CYP2A6 was placed under the control of the constitutive 35S promoter and transferred into Nicotiana tabacum via Agrobacterium-mediated transformation. Transgenic plants showed formation of the recombinant CYP2A6 enzyme but no obvious phenotypic changes. Unlike wild-type tobacco, the transgenic plants accumulated cotinine, a metabolite which is usually formed from nicotine in humans. This result substantiates that metabolic engineering of the plant secondary metabolism via mammalian P450 enzymes is possible in vivo. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Increased 24-hour energy expenditure in cigarette smokers.

We studied the effect of smoking on energy expenditure in eight healthy cigarette smokers who spent 24 hours in a metabolic chamber on two occasions, once without smoking and once while smoking 24 cigarettes per day. Diet and physical exercise (30 minutes of treadmill walking) were standardized on both occasions. Physical activity in the chamber was measured by use of a radar system. Smoking caused an increase in total 24-hour energy expenditure (from a mean value [+/- SEM] of 2230 +/- 115 to 2445 +/- 120 kcal per 24 hours; P less than 0.001), although no changes were observed in physical activity or mean basal metabolic rate (1545 +/- 80 vs. 1570 +/- 70 kcal per 24 hours). During the smoking period, the mean diurnal urinary excretion of norepinephrine (+/- SEM) increased from 1.25 +/- 0.14 to 1.82 +/- 0.28 micrograms per hour (P less than 0.025), and mean nocturnal excretion increased from 0.73 +/- 0.07 to 0.91 +/- 0.08 micrograms per hour (P less than 0.001). These short-term observations demonstrate that cigarette smoking increases 24-hour energy expenditure by approximately 10 percent, and that this effect may be mediated in part by the sympathetic nervous system. The findings also indicate that energy expenditure can be expected to decrease when people stop smoking, thereby favoring the gain in body weight that often accompanies the cessation of smoking.

Ablation of caterpillar labial salivary glands: technique for determining the role of saliva in insect-plant interactions.

There has been an ardent interest in herbivore saliva due to its roles in inducing plant defenses and its impact on herbivore fitness. Two techniques are described that inhibit the secretion of labial saliva from the caterpillar, Helicoverpa zea, during feeding. The methods rely on cauterizing the caterpillar's spinneret, the principal secretory structure of the labial glands, or surgically removing the labial salivary gland. Both methods successfully inhibit secretion of saliva and the principal salivary enzyme glucose oxidase. Caterpillars with inhibited saliva production feed at similar rates as the untreated caterpillars, pupate, and emerge as adults. Glucose oxidase has been suggested to increase the caterpillar's survival through the suppression of inducible anti-herbivore defenses in plants. Tobacco (Nicotiana tabacum) leaves fed on by caterpillars with ablated salivary glands had significantly higher levels of nicotine, an inducible anti-herbivore defense compound of tobacco, than leaves fed upon by caterpillars with intact labial salivary glands. Tomato (Lycopersicon esculentum) leaves fed upon by caterpillars with suppressed salivary secretions showed greatly reduced evidence of hydrogen peroxide formation compared to leaves fed upon by intact caterpillars. These two methods are useful techniques for determining the role that saliva plays in manipulating plant anti-herbivore defenses.

Effects of cigarette smoking and exposure to cadmium and lead on phenotypic variability of hepatic CYP2A6 and renal function biomarkers in men

Effects of cigarette smoking and exposure to dietary cadmium (Cd) and lead (Pb) on urinary biomarkers of renal function and phenotypic variability of cytochrome P450 2A6 (CYP2A6) were investigated in a group of 96 healthy Thai men with mean age of 36.7 year (19-57 years). In non-smokers, Cd burden increased with age (r = 0.47, P < 0.001). In current smokers, Cd burden increased with both age (r = 0.45, P = 0.01) and number of cigarettes smoked per day (r = 0.32, P = 0.05). Cd-linked renal tubular dysfunction was seen in both smokers and non-smokers, but Pb-linked glomerular dysfunction was seen in smokers only, possibly due to more recent exposure to high levels of Cd and Pb, as reflected by 30-50% higher serum Cd and Pb levels in smokers than non-smokers (P < 0.05). Exposure to dietary Cd and Pb appeared to be associated with mild tubular dysfunction whereas dietary exposure plus cigarette smoking was associated with tubular plus glomerular dysfunction. Hepatic CYP2A6 activity in non-smokers showed a positive association with Cd burden (adjusted P = 0.38, P = 0.006), but it showed an inverse correlation with Pb (adjusted beta = -0.29, P = 0.003), suggesting opposing effects of Cd and Pb on hepatic CYP2A6 phenotype. In contrast, CYP2A6 activity in current smokers did not correlate with Cd or Pb, but it showed a positive correlation with serum ferritin levels (r = 0.45, P = 0.01). These finding suggest that Pb concentrations in the liver probably were too low to inhibit hepatic synthesis of heme and CYP2A6 and that the concurrent induction of hepatic CYP2A6 and ferritin was probably due to cigarette smoke constituents other than the Cd and Pb. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Evidence for Concurrent Effects of Exposure to Environmental Cadmium and Lead on Hepatic CYP2A6 Phenotype and Renal Function Biomarkers in Non Smokers

We examined the interrelationships between phenotype of hepatic cytochrome P450 2A6 (CYP2A6), nephropathy, and exposure to cadmium and lead in a group of 118 healthy Thai men and women who had never smoked. Their urinary Cd excretion ranged from 0.05 to 2.36 mug/g creatinine, whereas their urinary Pb excretion ranged from 0.1 to 12 mug/g creatinine. Average age and Cd burden of women and men did not differ. Women, however, on average showed a 46% higher urinary Pb excretion (p < 0.001) and lower zinc status, suggested by lower average serum Zn and urinary Zn excretion compared with those in men. Cd-linked nephropathy was detected in both men and women. However, Pb-linked nephropathy was seen only in women, possibly because of higher Pb burden coupled with lower protective factors, notably of Zn (P < 0.001), in women compared with men. In men, Pb burden showed a negative association with CYP2A6 activity (adjusted beta = -0.29, p = 0.003), whereas Cd burden showed a positive association with CYP2A6 activity (adjusted beta = 0.38, p = 0.001), suggesting opposing effects of Cd and Pb on hepatic CYP2A6 phenotype. The weaker correlation between Cd burden CYP2A6 activity in women despite similarity in Cd burden between men and women is consistent with opposing effects of Pb and Cd on hepatic CYP2A6 phenotypic expression. A positive correlation between Cd-linked nephropathy (urinary N-acetyl-beta-D-glucosaminidase excretion) and CYP2A6 activity in men (r = 0.39, p = 0.002) and women (r = 0.37, p = 0.001) suggests that Cd induction of hepatic CYP2A6 expression and Cd-linked nephropathy occurred simultaneously.

A common biological basis of obesity and nicotine addiction.

Smoking influences body weight such that smokers weigh less than non-smokers and smoking cessation often leads to weight increase. The relationship between body weight and smoking is partly explained by the effect of nicotine on appetite and metabolism. However, the brain reward system is involved in the control of the intake of both food and tobacco. We evaluated the effect of single-nucleotide polymorphisms (SNPs) affecting body mass index (BMI) on smoking behavior, and tested the 32 SNPs identified in a meta-analysis for association with two smoking phenotypes, smoking initiation (SI) and the number of cigarettes smoked per day (CPD) in an Icelandic sample (N=34,216 smokers). Combined according to their effect on BMI, the SNPs correlate with both SI (r=0.019, P=0.00054) and CPD (r=0.032, P=8.0 × 10(-7)). These findings replicate in a second large data set (N=127,274, thereof 76,242 smokers) for both SI (P=1.2 × 10(-5)) and CPD (P=9.3 × 10(-5)). Notably, the variant most strongly associated with BMI (rs1558902-A in FTO) did not associate with smoking behavior. The association with smoking behavior is not due to the effect of the SNPs on BMI. Our results strongly point to a common biological basis of the regulation of our appetite for tobacco and food, and thus the vulnerability to nicotine addiction and obesity.

Maternal adipose tissue response to nicotine administration in the pregnant rat: effects on fetal body fat and cellularity

1. Nicotine has been implicated as a causative factor in the intrauterine growth retardation associated with smoking in pregnancy. A study was set up to ascertain the effect of nicotine on fetal growth and whether this could be related to the actions of this drug on maternal adipose tissue metabolism. 2. Sprague-Dawley rats were mated and assigned to control and nicotine groups, the latter receiving nicotine in the drinking-water throughout pregnancy. Animals were weighed at regular intervals and killed on day 20 of pregnancy. Rates of maternal adipose tissue lipolysis and lipogenesis were measured. Fetal and placental weights were recorded and analysis of fetal body water, fat, protein and DNA carried out. 3. Weight gains of mothers in the nicotine group were less in the 1st and 2nd weeks of pregnancy, but similar to controls in the 3rd week. Fetal body-weights, DNA, protein and percentage water contents were similar in both groups. Mean fetal body fat (g/kg) was significantly higher in the nicotine group (96.2 (SE 5.1)) compared with controls (72.0 (SE 2.9)). Rates of maternal lipolysis were also higher in the nicotine group. 4. The cause of these differences and their effects on maternal and fetal well-being is discussed.

Proteomic analysis of Porphyromonas gingivalis exposed to nicotine and cotinine

Cogo K, de Andrade A, Labate CA, Bergamaschi CC, Berto LA, Franco GCN, Goncalves RB, Groppo FC. Proteomic analysis of Porphyromonas gingivalis exposed to nicotine and cotinine. J Periodont Res 2012; 47: 766775. (c) 2012 John Wiley & Sons A/S Background and Objective: Smokers are more predisposed than nonsmokers to infection with Porphyromonas gingivalis, one of the most important pathogens involved in the onset and development of periodontitis. It has also been observed that tobacco, and tobacco derivatives such as nicotine and cotinine, can induce modifications to P. gingivalis virulence. However, the effect of the major compounds derived from cigarettes on expression of protein by P.gingivalis is poorly understood. Therefore, this study aimed to evaluate and compare the effects of nicotine and cotinine on the P.gingivalis proteomic profile. Material and Methods: Total proteins of P gingivalis exposed to nicotine and cotinine were extracted and separated by two-dimensional electrophoresis. Proteins differentially expressed were successfully identified through liquid chromatography-mass spectrometry and primary sequence databases using MASCOT search engine, and gene ontology was carried out using DAVID tools. Results: Of the approximately 410 protein spots that were reproducibly detected on each gel, 23 were differentially expressed in at least one of the treatments. A particular increase was seen in proteins involved in metabolism, virulence and acquisition of peptides, protein synthesis and folding, transcription and oxidative stress. Few proteins showed significant decreases in expression; those that did are involved in cell envelope biosynthesis and proteolysis and also in metabolism. Conclusion: Our results characterized the changes in the proteome of P.gingivalis following exposure to nicotine and cotinine, suggesting that these substances may modulate, with minor changes, protein expression. The present study is, in part, a step toward understanding the potential smokepathogen interaction that may occur in smokers with periodontitis.

Behavior of respiratory metabolism and pyridine nucleotide levels in Oxyrrhis marina during starvation

Máster en Oceanografía

Effects of chronic exposure to low-level cadmium on renal tubular function and CYP2A6-mediated coumarin metabolism in healthy human subjects

Relationships between cadmium (Cd) body burden, kidney function and coumarin metabolism were investigated using two groups of 197 and 200 healthy Thais with men and women in nearly equal numbers. A mean age of one group was 30.5 years and it was 39.3 years for the other group. Of 397, 20 subjects (5%) excreted urine Cd between 1.4 mug/g and 3.8 mug/g creatinine and these subjects faced 10-15% increase in the probability of having abnormal urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG-uria). The prevalence of NAG-uria varied with Cd body burden in a dose-dependent manner (chi(2) = 22, P < 0.008). Also NAG-nuria was one of the three kidney effect markers tested that showed the greatest strength of correlation with urine Cd in both men and women (r = 0.48 P < 0.001). In addition, urine Cd excretion of men and women showed a positive correlation (r = 0.46 to 0.54. P < 0.001) with urine 7-hydroxycoumarin (7-OHC) excretion which was used as a marker of liver cytochrome P450 2A6 (CYP2A6) enzyme activity. Urinary CA excretion accounted for 25% of the total variation in urine 7-OHC excretion (P < 0.001). These data suggest that Cd may increase the expression of CYP2A6 in liver, resulting in enhanced coumarin metabolism in subjects with high Cd body burden. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

Diagnosis, Treatment, And Follow-up Of Medullary Thyroid Carcinoma: Recommendations By The Thyroid Department Of The Brazilian Society Of Endocrinology And Metabolism.

Medullary thyroid carcinoma (MTC) originates in the thyroid parafollicular cells and represents 3-4% of the malignant neoplasms that affect this gland. Approximately 25% of these cases are hereditary due to activating mutations in the REarranged during Transfection (RET) proto-oncogene. The course of MTC is indolent, and survival rates depend on the tumor stage at diagnosis. The present article describes clinical evidence-based guidelines for the diagnosis, treatment, and follow-up of MTC. The aim of the consensus described herein, which was elaborated by Brazilian experts and sponsored by the Thyroid Department of the Brazilian Society of Endocrinology and Metabolism, was to discuss the diagnosis, treatment, and follow-up of individuals with MTC in accordance with the latest evidence reported in the literature. After clinical questions were elaborated, the available literature was initially surveyed for evidence in the MedLine-PubMed database, followed by the Embase and Scientific Electronic Library Online/Latin American and Caribbean Health Science Literature (SciELO/Lilacs) databases. The strength of evidence was assessed according to the Oxford classification of evidence levels, which is based on study design, and the best evidence available for each question was selected. Eleven questions corresponded to MTC diagnosis, 8 corresponded to its surgical treatment, and 13 corresponded to follow-up, for a total of 32 recommendations. The present article discusses the clinical and molecular diagnosis, initial surgical treatment, and postoperative management of MTC, as well as the therapeutic options for metastatic disease. MTC should be suspected in individuals who present with thyroid nodules and family histories of MTC, associations with pheochromocytoma and hyperparathyroidism, and/or typical phenotypic characteristics such as ganglioneuromatosis and Marfanoid habitus. Fine-needle nodule aspiration, serum calcitonin measurements, and anatomical-pathological examinations are useful for diagnostic confirmation. Surgery represents the only curative therapeutic strategy. The therapeutic options for metastatic disease remain limited and are restricted to disease control. Judicious postoperative assessments that focus on the identification of residual or recurrent disease are of paramount importance when defining the follow-up and later therapeutic management strategies.

Rootstocks induce contrasting photosynthetic responses of orange plants to low night temperature without affecting the antioxidant metabolism

Low temperatures negatively impact the metabolism of orange trees, and the extent of damage can be influenced by the rootstock. We evaluated the effects of low nocturnal temperatures on Valencia orange scions grafted on Rangpur lime or Swingle citrumelo rootstocks. We exposed six-month-old plants to night temperatures of 20ºC and 8ºC under controlled conditions. After decreasing the temperature to 8ºC, there were decreases in leaf CO2 assimilation, stomatal conductance, mesophyll conductance and CO2 concentration in the chloroplasts, in plant hydraulic conductivity and in the maximum electron transport rate driven ribulose-1,5-bisphosphate (RuBP) regeneration in plants grafted on both rootstocks. However, the effects of low night temperature were more severe in plants grafted on Rangpur rootstock, which also presented reduction in the maximum rate of RuBP carboxylation and in the maximum quantum efficiency of the PSII. In general, irreversible damage due to night chilling was found in the photosynthetic apparatus of plants grafted on Rangpur lime. Low night temperatures induced similar changes in the antioxidant metabolism, preventing oxidative damage in citrus leaves on both rootstocks. As photosynthesis is linked to plant growth, our findings indicate that the rootstock may improve the performance of citrus trees in environments with low night temperatures, with Swingle rootstock improving the photosynthetic acclimation in leaves of orange plants.

Repeated exposure of adolescent rats to oral methylphenidate does not induce behavioral sensitization or cross-sensitization to nicotine

Several lines of evidence indicate that the use of stimulant drugs, including methylphenidate (MPD), increases tobacco smoking. This has raised concerns that MPD use during adolescence could facilitate nicotine abuse. Preclinical studies have shown that repeated treatment with an addictive drug produces sensitization to that drug and usually cross-sensitization to other drugs. Behavioral sensitization has been implicated in the development of drug addiction. We examined whether repeated oral MPD administration during adolescence could induce behavioral sensitization to MPD and long-lasting cross-sensitization to nicotine. Adolescent male Wistar rats were treated orally with 10 mg/kg MPD or saline (SAL) from postnatal day (PND) 27 to 33. To evaluate behavioral sensitization to MPD in adolescent rats (PND 39), the SAL pretreated group was subdivided into two groups that received intragastric SAL (1.0 mL/kg) or MPD (10 mg/kg); MPD pretreated rats received MPD (10 mg/kg). Cross-sensitization was evaluated on PND 39 or PND 70 (adulthood). To this end, SAL- and MPD-pretreated groups received subcutaneous injections of SAL (1.0 mL/kg) or nicotine (0.4 mg/kg). All groups had 8 animals. Immediately after injections, locomotor activity was determined. The locomotor response to MPD challenge of MPD-pretreated rats was not significantly different from that of the SAL-pretreated group. Moreover, the locomotor response of MPD-pretreated rats to nicotine challenge was not significantly different from that of the SAL-pretreated group. This lack of sensitization and cross-sensitization suggests that MPD treatment during adolescence does not induce short- or long-term neuroadaptation in rats that could increase sensitivity to MPD or nicotine.

Aerobic and anaerobic metabolism for the zebrafish, Danio rerio, reared under normoxic and hypoxic conditions and exposed to acute hypoxia during development

In order to verify the influence of chronic and acute ambient oxygen levels from egg to adult stage of the zebrafish, in vivo oxygen consumption (MO2), critical tensions of oxygen (Pcrit), heart rate (fH) and total body lactate concentration (Lc) were determined for Danio rerio (Hamilton, 1822) raised at 28 °C under normoxic (7.5 mgO2.L-1 or 80 mm.Hg-1) and hypoxic conditions (4.3 mgO2.L-1) and exposed to acute hypoxia during different developmental stages. Our findings confirmed that very early stages do not respond effectively to ambient acute hypoxia. However, after the stage corresponding to the age of 30 days, D. rerio was able to respond to acute hypoxia through effective physiological mechanisms involving aerobic and anaerobic metabolism. Such responses were more efficient for the fishes reared under hypoxia which showed that D. rerio survival capability increased during acclimation to mild hypoxia. Measurements of body mass and length showed that moderate hypoxia did not affect growth significantly until the fish reached the stage of 60 days. Moreover, a growth delay was verified for the hypoxic-reared animals. Also, the D. rerio eggs-to-larvae survival varied from 87.7 to 62.4% in animals reared under normoxia and mild hypoxia, respectively. However, the surviving animals raised under moderated hypoxia showed a better aptitude to regulate aerobic and anaerobic capacities when exposed to acute hypoxia.

Influence of food components on lipid metabolism: scenarios and perspective on the control and prevention of dyslipidemias

Cardiovascular diseases (CVD) are the main causes of death in the Western world. Among the risk factors that are modifiable by diet, for reducing cardiovascular disease risks, the total plasma concentrations of cholesterol, triglycerides, LDL-C, and HDL-C are the most important. Dietary measures can balance these components of the lipid profile thus reducing the risk of cardiovascular diseases. The main food components that affect the lipid profile and can be modified by diet are the saturated and trans fats, unsaturated fats, cholesterol, phytosterols, plant protein, and soluble fiber. A wealth of evidence suggests that saturated and trans fats and cholesterol in the diet raise the total plasma cholesterol and LDL-C. Trans fats also reduce HDL-C, an important lipoprotein for mediating the reverse cholesterol transport. On the other hand, phytosterols, plant proteins, isoflavones, and soluble fiber are protective diet factors against cardiovascular diseases by modulating plasma lipoprotein levels. These food components at certain concentrations are able to reduce the total cholesterol, TG, and LDL-C and raise the plasma levels of HDL-C. Therefore, diet is an important tool for the prevention and control of cardiovascular diseases, and should be taken into account as a whole, i.e., not only the food components that modulate plasma concentrations of lipoproteins, but also the diet content of macro nutrients and micronutrients should be considered.