Cotinine

ENVIRONMENTAL TOBACCO SMOKE (ETS) is a term widely used to refer to the mixture of sidestream and exhaled mainstream smoke from tobacco products. ETS is of deep concern, as it pollutes the air in and around the locations where tobacco smoking occurs. The adverse health effects of active cigarette smoking have been investigated intensely subsequent to the mid-1900s. (1) Research on passive smoking (the inhalation of ETS by nonsmokers) began several decades ago, and there is now substantial evidence suggesting that passive smoking affects both children and adults and can cause malignant and nonmalignant diseases. (2-4) In the mid-1980s, ETS exposure from cigarette smoking was linked to an increased risk of lung cancer in nonsmokers. (5) Nonsmokers are exposed to ETS in the home, workplace, and any other location where smoking is not restricted.

Bidi is the Indian version of the cigarette, prepared by placing a small quantity of bidi tobacco (Nicotiana tobacum) on a dry temburni leaf (Diospyros melanoxy-Ion) and rolling the bidi by hand. This study monitored a group of bidi smokers and their female family members for biological effects from prolonged exposure to ETS from bidis. We examined the excretion rate of cotinine (a major metabolite of nicotine) among both active and passive bidi smokers and a nonexposed control group. We assessed the electrophilic burden imposed by ETS by estimating subjects' urinary excretions of thioether and glucuronides.

Materials and Method

We enrolled a total of 66 subjects for this study. Group 1 (n = 10) included subjects with no history of addiction to tobacco and no exposure to passive smoke (controls). Group 2 (n = 20) included male bidi smokers only (smokers). All smokers had no history of any addiction other than bidis. Groups 3 and 4 included the wives and daughters, respectively, of Group 2 members (passive smokers) (n = 25 wives and 11 daughters). Similarly, subjects in the passive smoker groups had no other addiction. All subjects belonged to a low socioeconomic group and resided in similar localities in the city of Ahmedabad, India. All subjects lived in poorly ventilated small rooms (approximately 1 m x 1.2 m x 1.2 m for each family member, with 1 door entrance). Demographic variables, anthropometric measurements, and information on the history of ETS exposure were recorded for all the subjects on precoded forms at the beginning of the study.

Sample collection. A 12-hr urine sample (750 ml) was collected from each subject, beginning with the sample after the early morning first void. After 12 hr, the samples were immediately brought to the laboratory and frozen without a preservative at -20[degrees]C until analysis.

Estimation of cotinine. Cotinine levels were estimated with the high-performance liquid-chromatography (HPLC) method described by Watson. (6) Three milliliters of each urine sample were placed in a conical centrifuge tube, and each sample was alkalinated with 5 M sodium hydroxide (NaOH), using 1% bromthymol blue as a pH indicator. An internal standard of 0.3 ml desmethylimipramine was added to each sample and mixed for 30 sec on a vortex mixer. The tubes were centrifuged to clear any emulsions, and 20 [micro]l were then injected into a Beckman HPLC injector (Beckman Instruments, Inc. [Berkeley, California]). The operating conditions were the same as described elsewhere. (6)

Estimation of thioethers. Each urine sample (5 ml) was acidified to pH 2.0 with 4 N hydrochloric acid (HCl), followed by extraction with 20 ml of ethylacetate. The organic layer was evaporated to dryness, and the residue was dissolved in 2 ml of distilled water. We added 0.5 ml of 4 N NaOH to 1 ml of the aforementioned extract, after which it was heated in a boiling water bath for 50 min. Hydrolysis was carried out under inert phase (i.e., in the presence of [N.sub.2]) in a screwcapped tube. After cooling to 0[degrees]C, 0.5 ml of 4 N HCI (0.65 M phosphate buffer, pH 7) was added to each tube. The reaction mixture comprised 2 ml of 0.5 M phosphate buffer (pH 7.1), 0.3 ml of 5,5'-dithio-bis-(1-nitrobenzoic acid) (DTNB) solution (0.4 mg DTNB/ml of 1% aqueous sodium citrate), and 0.25 ml of the neutralized hydrolysate. Readings were taken with an ultraviolet (UV) double spectrophotometer (Model U3210, Hitachi [Tokyo, Japan]). Corrections were made for urine color. Concentrations of thioether were calculated from a standard curve prepared with N-acetyl-Lcysteine.

Estimation of glucuronides. Urine samples (5 ml) were placed in a constant-temperature water bath with a shaker and allowed to react with 1 ml of 1% napththoresorcinol prepared in 95% absolute alcohol and 5 ml of concentrated HCl. The temperature of the reaction mixture was increased gradually, and finally maintained at the boiling point with the samples being shaken constantly. Once the reaction was completed, the mixture was brought to room temperature and extracted with an equal volume of ether. The presence of glucuronides was indicated by the appearance of a violetred color. Readings were taken with the Hitachi UV double-beam spectrophotometer. Sodium glucuronate was used as the standard for quantitative evaluation.

Statistical analysis. Student's t test and a modified t test were used to assess statistical significance. The modified t test was developed by Behrens-Fisher to account for samples with unequal variances; the table for determining statistical significance, using the modified t test, was created by Snedecor and Cochran. (7) Observations outside the mean [+ or -] 2.58 standard deviations (i.e., 1% of the observations) were classified as outliers and were excluded from the statistical analysis.

Chemicals. Cotinine, DTNB, N-acetyl-L-cysteine, and napththoresorcinol were purchased from Sigma Aldrich (St. Louis, Missouri). All other reagents used were of analytical grade.

Results

Age, gender, and anthropometric measurements for all subjects are presented in Table 1. All control and smoker subjects (Groups 1 and 2) were male. Five subjects in Group 2 dropped out of the study, citing personal reasons. All members of the passive smoking groups (Groups 3 and 4) were the female relatives (wives and daughters, respectively) of members of Group 2. Females are of interest as passive smokers in this study because they spend more time indoors than outdoors and, therefore, are assumed to be highly exposed.

Levels of urinary cotinine, thioethers, and glucuronides are given in Table 2. The mean cotinine level was significantly higher (p < 0.001) in smokers than among the other exposed groups; levels among the exposed were lowest in Group 4. Cotinine was not detected in any of the control subjects.

Urinary glucuronide excretion showed a nearly 2fold increase among active bidi smokers (98.83 [+ or -] 19.14 mmol/mol creatinine), compared with controls (51.91 [+ or -] 11.18). Glucuronide excretion levels were statistically significantly higher (p < 0.001) for Groups 2 and 3 (male smokers and their wives), compared with controls. They were somewhat higher for Group 4 (daughters: 57.79 [+ or -] 7.58 mmol/mol creatinine), compared with controls (51.91 [+ or -] 11.18), but this difference was not statistically significant.

There was a statistically significant incremental increase in thioether excretion among the exposed groups, compared with controls (p < 0.001). The greatest mean urinary thioether excretion was determined for Group 2 (smokers: 6.45 [+ or -] 2.73), followed by the female members of their families (Group 3 [wives] = 4.79 [+ or -] 1.48; Group 4 [daughters] = 4.85 [+ or -] 1.37). Means for thioether excretion for Groups 2 and 3 were significantly different from the mean for Group 1 (controls) (t test [p < 0.05]). Means for thioether excretion for Groups 2, 3, and 4 (i.e., all the exposed groups) were significantly different from the mean for Group 1 (controls) (modified t test [p < 0.001]). Intragroup comparisons revealed that differences between Groups 2 and 3, and Groups 3 and 4, were not significant (t test). Mean differences between Groups 2 and 3, however, were significantly different (modified t test [p < 0.05]).

We found that mean cotinine levels were correlated with means of glucuronides and thioethers. The correlations were statistically significant when all the exposed groups (Groups 2, 3, and 4) were aggregated into 1 exposed group. The correlation between urinary glucuronides and cotinine was r = .4966 (p < 0.0005); between thioethers and cotinine, r = .3015 (p < 0.03). These results indicate that levels of both glucuronides and thioethers increased with increased levels of cotinine.

The correlation between log glucuronide and log cotinine was observed as r = .5845 (p < 0.0005). This result indicates that the glucuronide-cotinine relationship (regression) follows a power function (i.e., in the form of y = [a][[x.sup.b]]). From the present data, the equation is: glucuronides = 73.091 x (cotinine) (0.177), cotinine > 0 (Fig. 1). Detailed regression constants are given in Table 3.

[FIGURE 1 OMITTED]

Discussion

In the present investigation, we evaluated 3 parameters (i.e., cotinine, thioether, and glucuronides) and their relationship with ETS. Subjects included a group of male active bidi smokers, 2 groups of female passive bidi smokers related to the male subjects, and a group of nonexposed male controls. We determined a positive relationship between the 3 parameters and exposure to ETS from bidis.

Human exposures to hazardous chemical mixtures are ascertained by the demonstration of a specific component or its metabolites in biological samples. In this study, we used nonspecific indicators, such as urinary thioethers and glucuronides, to denote exposure to genotoxic agents. Cotinine was used as an indicator for exposure to a tobacco-specific alkaloid: nicotine. Cotinine was selected for its tobacco specificity and its long-term biological half-life relative to nicotine. Cotinine was absent in the urine samples of controls, but it was present in urine obtained from subjects in all the exposed groups (i.e., both active and passive bidi smokers). The mean urinary cotinine was 4- to 8-fold higher for active bidi smokers than for both groups of passive smokers. The elevated cotinine excretion among nonsmoking wives appeared related to considerable nicotine absorption from bidi tobacco smoke in small, poorly ventilated rooms.

Many hazardous chemicals are metabolized into reactive electrophilic forms by the human body. These electrophilic forms can then interact with cellular macromolecules to produce genotoxicity. Glutathione and glucuronic acid prevent this hazardous interaction by nonspecifically conjugating with many electrophiles. The substances are then excreted in urine as thioethers and glucuronides, respectively. We assessed the electrophilic burden imposed by ETS from bidi tobacco by estimating urinary thioether and glucuronide levels among several categories of exposed subjects and controls.

Urinary thioethers increase upon exposure to alkylating agents and electrophilic chemicals. (8-11) In addition, concentrations are higher among consumers of highprotein diets. (12) Given that all the subjects in our study were vegetarians, the results could not have been confounded by a high-protein diet.

Tomatoes and several vegetables, such as potatoes and cauliflower, contain nicotine, (13) but they provide a low level of nicotine exposure compared with that posed by ETS. A preliminary inquiry revealed that our subjects consumed these types of vegetables. Given that the subjects belonged to a low socioeconomic group, they could not afford to buy costlier vegetables. As nicotine is a minor alkaloid in these vegetables, we did not consider them to be a serious secondary source of nicotine.

Other factors that reportedly increase urinary thioether excretion are occupational exposures to genotoxic chemicals, such as those used in the rubber industry, in chemical plants, and by asphalt workers. Whereas the excretion of both urinary thioethers and glucuronides is nonspecific for verifying exposures to genotoxic agents, we investigated both parameters to determine exposure to chemical mixtures such as ETS from bidis. The exposed groups showed significantly elevated levels of urinary thioethers. We also found that the excretion of glucuronides--an index to the exposure to genobiotics (14)--showed a good correlation with cotinine.

At present, cotinine is the most specific and sensitive biomarker for exposure to nicotine from ETS. Its limitation is that it indicates ongoing exposure, not long-term exposure. Conversely, nicotine-derived nitrosoamines, such as 4-(methylnitrosoamine)-1-(3-pyridyl)-1-butanone, are specific for tobacco exposure and are metabolized to a butanol metabolite 4-(methylnitrosoamine)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronide (NNAL-GLUC). (15) Urinary levels of NNAL and NNALGLUC are elevated in nonsmokers who are exposed to ETS. In one small study, the correlation between urinary NNAL + glucuronide and urinary cotinine levels was strong. (13) In the present investigation, we did not measure urinary NNAL + glucuronide, but we observed exposure-related increments of glucuronide in the urines of both active and passive bidi smokers.

ETS is a common indoor air pollutant that has been implicated in the development of various respiratory illness in children. (16) Two elaborate studies on ETS--Jenkins et al. (17) (16 cities) and Hammond et al. (18) (25 work sites)--offer information on indoor ETS exposures. There is a large body of epidemiological evidence associating ETS with childhood respiratory diseases, (16) and with adult lung cancer (19) and heart diseases. (20) We found significant levels of the measured parameters among passive smokers, primarily because the females in our study spent much time indoors in small, poorly ventilated spaces. Our study, as well as prior studies on occupational health problems among non-Virginia tobacco handlers, (21-23) highlight the hazards of tobacco exposure, especially for passive smokers in a low socioeconomic group who live in poorly ventilated rooms where the chance of exposure to ETS is great. Our findings confirm that bidi smoking is a health concern not only for active smokers, but also for the passive smoking members of their households.

References

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Submitted for publication August 29, 2001; revised; accepted for publication August 5, 2002.

Requests for reprints should be sent to Dr. S. K. Ghosh, Deputy Director, National Institute of Occupational Health, Meghani Nagar, Ahmedabad 380 016, India.

E-mail: sandip_nioh@yahoo.co.in

SANDIP K. GHOSH

VIJAY K. BHATNAGAR

PANKAJ B. DOCTOR

MAHESH P. SHAH

RAJNIKANT J. AMIN

PRADIP K. KULKARNI

National Institute of Occupational Health

Indian Council of Medical Research

Meghani Nagar

Ahmedabad, India

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