THE EFFECTS OF TOBACCO SMOKE EXPOSURE ON CAFFEINE METABOLISM

Abstract

Caffeine and nicotine are some of the most often self-administered substances worldwide. Very often they are taken simultaneously and it seems that this fact is correlated with the amount of caffeine and nicotine administered. The aim of this study is to determine, whether tobacco smoke influences the metabolism of caffeine. The secondary task is to establish whether caffeine has an effect on elimination of cotinine, nicotine's main metabolite. The results showed that tobacco smoke influences the metabolism of caffeine by accelerating its elimination, by the means of induced CYP1A2 activity. As far as cotinine is concerned, no influence of caffeine on its elimination was observed. [PUBLICATION ABSTRACT]

Headnote

ABSTRACT

Caffeine and nicotine are some of the most often self-administered substances worldwide. Very often they are taken simultaneously and it seems that this fact is correlated with the amount of caffeine and nicotine administered. The aim of this study is to determine, whether tobacco smoke influences the metabolism of caffeine. The secondary task is to establish whether caffeine has an effect on elimination of cotinine, nicotine's main metabolite. The results showed that tobacco smoke influences the metabolism of caffeine by accelerating its elimination, by the means of induced CYP1A2 activity. As far as cotinine is concerned, no influence of caffeine on its elimination was observed.

- Keywords: caffeine, cotinine, metabolism, tobacco smoke -

INTRODUCTION

Caffeine and nicotine are some of the most commonly self-administered drugs worldwide. Caffeine is the vital compound found in coffee and energizing beverages, whereas nicotine is the main psychoactive substance in tobacco and tobacco smoke. The daily intake of caffeine varies worldwide, as it depends on the type of beverage that is consumed. According to a projection made by the Food and Agriculture Organization (FAO), consumption of caffeinated beverages in 2010 is forecasted to increase (FAO, 20 10).

Caffeine affects the respiratory, cardiovascular and, most notably, central nervous system (CNS). As far as the cardiovascular effect is concerned, caffeine both decreases heart rate and increases blood pressure (KOURTIDOU-PAPADELI et ed., 2002).

Caffeine's most important metabolites are: theobromine (3,7-dimethylxanthine), paraxanthine (1,7-dimethylxanthine) and theophylline (1,3-dimethylxanthine). Enzymes responsible for caffeine metabolism are cytochrome P450 isoforms (Fig. 1).

Cytochrome CYP1A2 is mainly involved in transformation of caffeine to paraxanthine, but also to theophylline and theobromine, to some extent. CYP2E1 metabolizes caffeine to theobromine and theophylline (ARNAUD, 201 1).

Tobacco smoke is the product of tobacco combustion. There are two kinds of tobacco smoke: mainstream smoke, inhaled by the smoker, and side-stream smoke, emitted by the cigarette between puffs (MAURER and SYRIGOS, 2006). The smoke that remains suspended in the air is called Environmental Tobacco Smoke (ETS). The most frequently used biomarker of tobacco smoking and exposure to ETS is cotinine, the metabolite of nicotine, which is also a biomarker of ETS (PRIGNOT, 201 1).

Tobacco smoke contains over 4,300 different compounds, among them many established carcinogens, which results in increased risk of developing various forms of cancer, e.g. lung cancer (MAURER and SYRIGOS, 2006). WHO study reports that the highest prevalence of tobacco use is in Europe, North America and Australia (WHO, 2011).

As mentioned before, tobacco smoke contains various substances that can interact with administered drugs, among others with caffeine and its metabolism (ZEVIN and BENOWITZ, 1999). Polycyclic aromatic hydrocarbons induce cytochrome P450 activity which may result in rapid caffeine metabolism. Furthermore, nicotine was believed to induce CYP1A2 activity (ZEVIN and BENOWITZ, 1999), recent studies shown relation between nicotine induction of the cytochrome P450 isoform and caffeine metabolism (HUKKANEN et ed., 201 1). However, other components of tobacco smoke, such as heavy metals and carbon monoxide are known inhibitors of the cytochrome P450 (ZEVIN and BENOWITZ, 1999).

According to findings of a study concerning the interaction between caffeine and tobacco smoke published in 1989 (BROWN and BENOWITZ, 1989), the cigarette consumption is greater during caffeine intake and the concentration of nicotine is higher during low-dose of caffeine compared with no-caffeine intake. It also seems that smokers drink more coffee, however, there are also reports stating that caffeine consumption had practically no influence on nicotine intake (TANDA and GOLDBERG, 2000). According to another paper, although coffee- drinking smokers smoked more than non-coffee drinkers, the number of cigarettes smoked was uncorrelated to the type of beverage drank (caffeinated or non-caffeinated) (BLANK et ed., 2007). Moreover, smokers drinking non-caffeinated beverages smoked significantly more than non-drinking smokers.

The aim of this study was to describe the effect of exposure to tobacco smoke on caffeine metabolism in animal model.

MATERIALS AND METHODS

Reagents

Caffeine, theophylline, theobromine and paraxanthine, as well as cotinine in the form of crystal powder were obtained from Sigma- Aldrich, NJ, USA, and 8-bromotheophylline also in the form of crystal powder was synthesized in the Department of Organic Chemistry, Faculty of Chemistry of the Jagiellonian University, Krakow, Poland. Stock solutions (1 mg/mL) of the analytes were prepared in water. Working solutions at following concentrations: 0, 10 (only for cotinine), 50, 100, 200, 500, 1,000, 2,000 and 5,000 ng/mL were prepared in water by appropriate dilution of the stock solution. Concentration of internal standard (8-bromotheophylline) in water was 50 µL/toL.

Sample preparation

Firstly, acidic extraction was performed: 200 µ?> of a phosphate buffer (pH 3) and 20 [iL of internal standard (8-bromotheophylline) were added to 500 [iL of rat serum or to 500 µ? of standard solution (when preparing calibration curve) and mixed on a Vortex. To each test tube 3 mL of chloroform (Sigma, ? PLC Grade) was added and solutions were shaked for 15 min and centrifuged for 20 min at 3,000 rpm. The organic phase was separated (2 mL) and evaporated at 40°C in nitrogen stream. The dry residue was dissolved in 300 µL of mobile phase.

The process of cotinine extraction was carried out using the same method as caffeine and its metabolites (theobromine, theophylline and paraxanthine).

Chromatographic condition

100 µL of solution was injected into the chromatographic column (Supelcosil LC-8, 25 cm ? 4.6 mm, 5 µ??). A diode-array detector (Merck Hitachi L-3000 Photo Diode Array Detector) and a UV-spectrophotometer (275 nm) (Spectra 1 00 Therma Seperation Products) were used simultaneously. The DAD-detector was used for higher concentrations and the UV-detector for lower ones. Cotinine samples were measured at 254 nm.

For the analysis the isocratic elution was applied. The flow of the mobile phase (1.19 g of sodium octanesulfonate and 4.54 g of potassium hydrogen phosphate in 880 mL of deionised water and 120 mL of acetonitrile (Sigma, HPLC Gradient Grade)) was 1.1 mL/min and constant.

The retention times of the analytes were as follows: theobromine 7,6 min, paraxanthine 12.1 min, theophylline 12.5 min, caffeine 25.6 min and that of 8-bromotheophylline was 28.8 min. Cotinine's retention time was 9. 1 min.

Animal experiment

One hundred and five Wistar rats (250-300 g) bred at the Department of Toxicology of the Poznan University of Medical Sciences were housed in polycarbonate cages containing sawdust bedding. A standard pellet diet and water were available ad libitum. The 12/12 h light/ dark cycle, temperature 20°-22°C and humidity 50-60% were maintained throughout the study. After 14 days of acclimatization, the rats were randomized and divided into three groups of 45 animals each. The first group of rats was given caffeine intraperitoneally in a dose of 10 mg/kg of body weight. Animals from the second group animals were exposed to tobacco smoke for 6 h per day, for 5 days. CO concentration was taken as an index of tobacco smoke concentration and was maintained at 1,500 mg CO/m3. Rats were exposed in a dynamic toxicological chamber (FLOREK and MARSZALEK, 1999) to tobacco smoke generated from a Polish brand of cigarettes without a filter tip. Animals from the third group were exposed to tobacco smoke at a concentration of 1,500 mg CO/m3 of air during 5 days, 6 hours a day and after the last day of exposure, caffeine was injected intraperitoneally in the dose of 1 0 mg/kg body weight. After exposure to tobacco smoke (second group) or administration of caffeine (first and third group), animals were anesthetized (Xylocaine 40 mg/kg and ketamine 5 mg/kg). Blood samples were collected at nine time-points (15 and 30 min, and 1, 2, 3, 6, 9, 12 and 18 h) with three rats per point.

After blood clotting, the samples were centrifuged and the obtained serum was kept at a temperature of -18°C until analysis was performed (no longer than two weeks). The protocol for this animal experiment was approved by the Local Ethics Commission for Animal Studies in Poznan.

Statistic and pharmacokinetic calculation

In our study analysis of variances was applied for statistic data. Furthermore, all pharmacokinetic parameters, that is: area under the curve (AUC), area under the first moment curve (AUMC), clearance (CI; calculated only for caffeine), volume of distribution (V; calculated only for caffeine), mean residence time (MRT), elimination rate constant (k; calculated for caffeine and cotinine), biological half-time (t1/2) were calculated by the means of the statistical moment analysis. To calculate the parameters we used the non-compartmental model.

RESULTS

In our studies paraxanthine, theophylline and theobromine were used as markers for influence of tobacco smoke on caffeine metabolism and cotinine as an indicator of exposure to tobacco smoke.

Due to the analytical aspect of the paper, Table 1 contains analytical parameters of our study.

The lowest concentration on the calibration curve was taken as the limit of quantitation, whereas the limit of detection was calculated as three times lower as the limit of quantitation. Linearity is defined as the concentration range was the concentration is directly proportional to the analytical signal - in the case of our study the area under the peak.

Tables 2 and 3 display the values of pharmacokinetic parameters of caffeine and its metabolites (Table 2), as well as of cotinine (Table 3).

The pharmacokinetic parameter describing exposure to tobacco smoke (area under curve of caffeine) has a lower value in the group exposed to tobacco smoke and caffeine comparing to animals that had only caffeine administered.

Furthermore, mean residence time and biological half-time (twofold lower) as well as clearance and the elimination rate constant (twotimes higher), depict a faster elimination after exposure to tobacco smoke. No change in volume of distribution indicates lack of influence of tobacco smoke on caffeine distribution throughout the organism. The faster elimination of caffeine due to tobacco smoke exposure is also depicted in Fig. 2.

Caffeine metabolism is catalyzed by cytochrome P450 isoforms: CYP2E1 and CYP2A1 (ARNAUD, 2011). Acceleration of caffeine elimination after tobacco smoke exposure is mainly caused by polycyclic aromatic hydrocarbons which are known to induce cytochrome P450 isoforms (VILLARD et ed., 1998; ZEVIN and BENOWITZ, 1999).

Paraxanthine is caffeine's main metabolite in man (ARNAUD, 201 1). According to data collected in Table 2, area under concentration-time curve is the only pharmacokinetic parameter for paraxanthine that is not statistically different for tobacco and non-tobacco smoke exposed groups. Mean resident time and biological half-time decreased 1 .5-time after exposure to tobacco smoke, whereas elimination rate constant increased 1. 5-time. The faster elimination of par - axanthine is shown in Fig. 3. The highest concentrations were in the third hour for both with and without tobacco smoke exposure.

The metabolite which is of high medical importance, as it is used for asthma treatment, is theophylline. All its pharmacokinetic parameters were statistically different after tobacco smoke exposure. Theophylline highest concentrations were in the sixth hour and second hour for rats exposed only to caffeine and for rats exposed to tobacco smoke, respectively. Exposure to tobacco smoke accelerates the process of caffeine's biotransformation to theophylline (Fig. 3). Moreover, its elimination is more rapid, which is confirmed by the two times higher elimination rate constant as well as statistically significantly lower values of MRT and biological half-time. Area under curve decreased in half (Table 2).

For theobromine the concentration-time profile (Fig. 3) and all pharmacokinetic parameters (Table 2) were similar.

In both experimental groups the maximum concentration of cotinine was achieved 30 min after end of exposure. Calculated pharmacokinetic parameters of cotinine in group exposed to tobacco smoke and in the group exposed to smoke and caffeine indicated that caffeine cotreatment does not influence cotinine elimination (Table 3). None of the parameters describing rate of elimination (biological half-lives, elimination rate constants and mean resident times) differ statistically.

DISCUSSION

According to our research, caffeine has no influence on nicotine's metabolism, since there were no changes in pharmacokinetic parameters describing a xenobiotic's elimination rate after co-administering of caffeine and nicotine. This is supported by a paper published by Kozlowskl (1976), who found that smokers absorb more nicotine while not drinking coffee. Similarly, in a different paper can be found that caffeine had no influence on nicotine intake (CHAIT and GRIFFITHS, 1983). However, a different study (MARSHALL et al, 1980) reports that coffee drinking did increase the number of smoked cigarettes, although they observed this effect also for water and a non-caffeinated drink which may suggest that the increase of nicotine consumption is not solely connected to caffeine's pharmacologic effects.

Furthermore, our study revealed that tobacco smoke does increase caffeine's elimination rate as well as its two metabolites: theophylline and paraxanthine. Pharmacokinetic parameters for theobromine (Table 2) did not differ after exposure to tobacco smoke and this suggests that tobacco smoke does not alter its elimination rate

Changes in caffeine's metabolism due to tobacco smoke exposure is also described in MARSHALL et al (1980). According to their findings coffee drinkers smoke more than non-drinkers. In a different study (DE LEON et al., 2003) it is reported that smokers had lower plasma caffeine concentration than non-smokers which also supports our conclusion that tobacco smoke increases caffeine metabolism.

Paraxanthine, theophylline and theobromine are caffeine's metabolites, all catalysed by cytochrome P450 isoforms. The first one, only by CYP1A2, and the rest mainly by CYP2E1 and partially CYP2A1 (ARNAUD, 2011). One of the tobacco smoke constituents is polycyclic aromatic hydrocarbons which induces cytochrome P450 isoforms, mainly CYP1A2 (KROON, 2007).

According to data collected during the experiment, tobacco smoke increases elimination of caffeine (Table 2 and Fig. 2), as that of paraxanthine and theophylline (Table 2 and Fig. 3).

In the sixth hour, paraxanthine/caffeine ratios as well as the theophylline/caffeine ratio for the tobacco-smoke exposed group are over fourtimes higher than in the group exposed only to caffeine (Fig. 4). In the case of theobromine, the ratio comparison shows tobacco-smoke exposed group ratio to be over 5.5-times higher than for the caffeine-exposed one (Fig. 4).

The comparison of the group exposed to tobacco smoke and both tobacco smoke and caffeine, after nine hours, reveals that theobromine/caffeine and paraxanthine/caffeine ratios are similar. Theophylline/caffeine ratio of the group exposed to both factors (i.e. tobacco smoke and caffeine) is twofold lower than the ratio of the group exposed only to caffeine.

In our study we observed that paraxanthine and theophylline are metabolized faster than theobromine. A similar study that investigated the effect of rutaecarpine, which is also a CYP450 inducer, shows an increase of elimination rate of all caffeine metabolites after exposure to this drug (NOH et al., 2011). However, according to our findings, tobacco smoke, which contains CYP450 inducer, such as polycyclic aromatic hydrocarbons, has no such effect on theobromine (Table 2). The tobacco smoke related cytochrome induction can serve as an explanation. CYP450 isoforms involved in caffeine, as well as paraxanthine and theophylline, metabolism are induced by polycyclic aromatic hydrocarbons present in tobacco smoke, whereas those connected to theobromine biotransformation seem not to be altered. Caffeine metabolism is accelerated, theobromine metabolism does not change, therefore, theobromine/caffeine ratio is high.

As mentioned before, the reason for this is the presence of polycycllc aromatic hydrocarbons in tobacco smoke. These compounds of tobacco smoke induce several of CYP450 isoforms, including CYP1A2, which is not only responsible for caffeine metabolism but also the metabolites' (paraxanthine and theophylline) biotransformation to their respective metabolites (ARNAUD, 201 1). CYP1A2 catalyses theophylline transformation to both 1-methylxanthine and 3-methylxanthine. Therefore, higher activity of this enzyme causes faster elimination, via biotransformation, of caffeine as well as paraxanthine and theophylline.

CONCLUSION

The aim of the study was to determine if tobacco smoke influences caffeine's metabolism and caffeine's influence on nicotine metabolism using cotinine as a marker. No changes in pharmacokinetic parameters describing rate of elimination of cotinine indicate lack of caffeine's influence on the metabolism of nicotine. However, exposure to tobacco smoke does accelerate caffeine elimination. Our study demonstrates that tobacco smoke significantly increases caffeine elimination, hence its lower concentration in the organism. Tobacco smoke also influences elimination of two caffeine metabolites - theophylline and paraxanthine, whereas pharmacokinetic parameters for theobromine are unchanged after tobacco smoke exposure.

These data can serve as an explanation for the observed phenomenon of smokers drinking more coffee than non-smokers.

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Paper received February 15, 2012 Accepted August 27, 2012