Antibodies to photoproducts of deoxyribonucleic acids irradiated with ultraviolet light.

A rabbit immunized with complexes of methylated bovine serum albumin and ultraviolet-irradiated DNA from calf thymus produced antibodies directed toward the photoproducts in the DNA. Serologic activity appeared after irradiation of DNA at 270 mmicro and decreased upon irradiation at 235 mmicro. The antigenic determinants of the ultraviolet-treated DNA appear to be photoproducts associated primarily with thymine, as measured by direct dependence of serologic activity on the adenine-thymine content of the DNA, and by inhibition of the Serlolgic reaction by the irradiated di-,tri-,and tetra-(thymidine-5'-phosphate nucleotides.

Aflatoxin B; specific antibodies and their use in radioimmunoassay.

New Zealand White rabbits immunized with covalent conjugates prepared from polylysine and the O-carboxy-methyloxime derivatives of either aflatoxin B1 (AFB1) or an analogue, 5,7-dimethoxycyclopentenon (2,3-c) coumarin, produced antibodies that bind 3H-AFB1. The specificities of the antisera with respect to aflatoxins BI, B2a, G1, G2, Q1, P1, and some other structually related compounds were determined. Radioimmunoassays that can detect levels as low as 0.27 pmoles (0.06 ng) of AFB1-were used to analyze serum, urine, and crude extracts of corn and peanut supplemented with aflatoxin. In the foodstuffs, as little as 1 mug AFB1/kg was measured. The immunoassay was at least as sensitive and specific as other available analytic methods, but did not require the purification of samples by chromatography before analysis. The technique may be particularly useful in epidemiologic studies designed to study the possible relationship between chronic aflatoxin ingestion and cancer.

Plasma nicotine and cotinine concentrations in habitual smokeless tobacco users.

Plasma nicotine and cotinine levels were measured in habitual users of smokeless tobacco. The subjects were 12 male college students who regularly used smokeless tobacco (11 dipped snuff and one chewed tobacco) and did not smoke cigarettes. Subjects abstained from tobacco use overnight and blood was drawn at 8 A.M. and again after a single day of ad libitum consumption of their own tobacco product. Subjects recorded the times at which tobacco was used and the remainder product was weighed. Plasma samples were analyzed by both gas-liquid chromatography (GLC) and radioimmunoassay (RIA) techniques. Subjects consumed about one third of a can of moist ground snuff (10.8 gm) in eight dips spaced throughout the day. Nicotine absorption was observed and an increase in mean plasma concentration fro 2.9 ng/ml after overnight abstinence to 21.6 ng/ml after 6 to 8 hr ad libitum consumption was recorded. Plasma cotinine concentrations rose from a morning mean of 137.3 ng/ml to an afternoon mean of 197.2 ng/ml, concentrations that are typical of those reached in regular cigarette smokers. Subjects fell into two subgroups by post hoc analysis: two-thirds absorbed substantial amounts of nicotine and one-third appeared to have almost no absorption. Subjective effects of tobacco use were not marked; there was little perception of physiologic changes, stimulation, or feelings of relaxation/satisfaction. Results are discussed in terms of pharmacologic effects, comparison of results from GLC and RIA methodologies, and implications for health behaviors.

Plasma adriamycin and daunorubicin levels by fluorescence and radioimmunoassay.

In 38 adriamycin experiments and 4 daunorubicin experiments, radioimmunoassay readily and reproducibly detects and estimates these drugs and immunologically similar metabolites in patients' plasma and urine to at least 120 hr after dosing without interference by concurrent medication. The plasma drug decay follows first-order kinetics in a triphasic pattern. Radioimmunoassay and fluorescence assay show similar decay up to 4 hr but diverge at that point with the fluorescence assay yielding higher values. Pharmocokinetic differences are amplified in patients with liver dysfunction and may be caused by fluorescent drug metabolites not sensitive to radioimmunoassay or nonspecific fluorescent materials. The radioimmunoassay offers the capability to measure adriamycin and daunorubicin in clinical settings in which fluorescence assay is not available.

Antibodies and radioimmunoassays for phosphoserine, phosphothreonine and phosphotyrosine. Serologic specificities and levels of the phosphoamino acids in cytoplasmic fractions of rat tissues.

For antibody production, the O-phosphorylated derivative of tyrosine, threonine, or serine was covalently linked to succinylated bovine albumin via the carbodiimide reaction. Each conjugate was then complexed with methylated bovine albumin for immunization of rabbits. To determine binding, the corresponding O-phosphorylated [3H]amino acids were chemically synthesized. In addition, these 3H-phosphorylated derivatives were acylated (with succinic or acetic anhydride) to obtain ligands whose structures resemble those present in the immunogen. The acylated ligands bound to their respective antibodies more effectively: in some cases binding was about three orders of magnitude greater than their non-acylated counterparts. Radioimmunoassays were therefore developed using the N-succinyl-[3H]phosphoamino acids. When the unlabeled N-succinyl-phosphorylated amino acids were used as inhibitors in the homologous immune systems, 50% displacement of the labeled ligand was found with 0.06, 0.27 or 0.8 pmol of the tyrosine, threonine, or serine derivative, respectively. The antibodies were highly specific for the homologous hapten; the requirement for the phosphate group on the acylated amino acid was essentially absolute. Antibody content (expressed as mg/ml serum) and apparent binding constants for the N-succinyl derivatives in individual bleedings of immune sera were 1.9 and 1 X 10(10) M-1 for phosphotyrosine, 0.825 and 6 X 10(8) M-1 for phosphothreonine, and 0.150 and 2 X 10(8) M-1 for phosphoserine. The radioimmunoassays were used to quantitate the phosphoamino acids in cytoplasmic fractions of rat tissue extracts. The production of antibodies to phosphorylated O-tyrosine has been reported previously, but to our knowledge, this represents the first report of antibodies specific for O-phosphorylated serine and threonine residues.

Stereospecific monoclonal antibodies to nicotine and cotinine and their use in enzyme-linked immunosorbent assays.

Stereospecific monoclonal antibodies (McAb) have been prepared against the tobacco alkaloid (S)-(-)-nicotine and its major metabolite (S)-(-)-cotinine. Nine anti-nicotine and 4 anti-cotinine hybridomas, selected by a screening procedure that utilized immunoprecipitation of the 3H-labeled natural isomers of nicotine or continine, were grown in the ascites fluid of pristane-primed syngeneic BALB/c mice. Antibodies in concentrations up to 7.5 mg/ml ascites and with binding affinities that generally exceeded 10(8) M-1 were obtained. Enzyme-linked immunosorbent assays (ELISAs) were developed in which nicotine or cotinine derivatives bound covalently to poly-L-lysine were coated onto wells of polyvinyl chloride microtiter plates. Coated wells were incubated sequentially with McAb in the presence or absence of inhibitor, rabbit anti-mouse immunoglobulin, then horseradish peroxidase-labeled protein A (HRP-SpA) before addition of substrate. The antibodies are highly specific and show minimal cross-reactivity with several nicotine metabolites and other structurally related compounds. In the respective assays, only 0.25 ng (S)-(-)-nicotine and 0.12 ng (S)-(-)-cotinine are required to give 50% inhibition of antibody binding, and as little as 0.05 ng nicotine and 0.02 ng cotinine give 15% inhibition. These assays are 5-10 times more sensitive than analogous ELISAs developed with rabbit antisera and HRP-SpA or conventional radioimmunoassays (RIAs) that utilize the rabbit antisera and 3H-labeled ligands. There was good correlation between the levels of nicotine (r = 0.967) and cotinine (r = 0.981) found in saliva samples from smokers and non-smokers assayed by McAb-based ELISAs and conventional RIAs.

The effect of nicotine on the delay of gastric emptying.

The effect of cigarette smoking and its active component, nicotine, on the gastric emptying of solid food was assessed in a randomized double-blind crossover design. Ten regular smokers were studied after a 6 h fast and least 18 h after their last cigarette. Subjects smoked a total of three high (1.91 mg) or low (0.17 mg) nicotine cigarettes, before and after a technetium-labelled solid meal and were scanned by gamma camera periodically over a 2-h period. All calculations of gastric emptying revealed a significant delay after smoking high versus low nicotine cigarettes in: mean per cent isotope remaining in the stomach at each measurement point from 90-120 min; amount of meal remaining in the stomach at 2 h; and mean time at which 50% of the meal had emptied (T1/2). Delay in gastric emptying was significantly correlated with increase in serum nicotine concentration on the high nicotine day.

Maternal smoking during pregnancy, urine cotinine concentrations, and birth outcomes. A prospective cohort study.

BACKGROUND: Most studies of the reproductive consequences of cigarette smoking base exposure on self-reported smoking habits. This study examines the relationship of birth outcomes to the timing and intensity of maternal active and passive smoking estimated both from self-reports and from cotinine concentration in maternal urine during early, middle, and late gestation. METHOD: This cohort study included 740 white and Hispanic women who obtained antenatal care at the East Boston Neighborhood Health Center between 1986 and 1992. At each antenatal visit, information on maternal active and passive smoking was obtained by a detailed questionnaire, and by measurement of urine cotinine concentrations. Infant birth outcomes were obtained from hospital records. Multiple linear regression was used to evaluate antenatal smoking variables on birth outcomes, with adjustment for maternal demographic characteristics, reproductive history, alcohol use, maternal weight and height, and infant gender. RESULTS: The percentage of mothers who ever smoked cigarettes during pregnancy was 55.5% for white and 10.2% for Hispanic women. A significant inverse exposure-response relationship between cotinine concentration in maternal urine and infant size at birth was demonstrated. However, the relationship was less clear between maternal self-reported smoking status and these outcomes. For the entire gestation, a 1000 ng increase in mean urine cotinine concentration was associated with a 59 +/- 9 g reduction in birthweight, a 0.25 +/- 0.05 cm reduction in length, and a 0.12 +/- 0.03 cm reduction in head circumference, respectively. For maternal passive smoking, the much smaller magnitude of effect precludes firm conclusions. CONCLUSIONS: These data suggest that preventing and reducing active maternal smoking during pregnancy may have a beneficial impact on infant size at birth.

Nicotine from cigarette smoking increases circulating levels of cortisol, growth hormone, and prolactin in male chronic smokers.

Results of this study indicate that nicotine from cigarette smoking increases circulating levels of cortisol, growth hormone, and prolactin in male chronic smokers. Previous studies have not addressed the question of whether the stimulus for smoking-related hormone release is the 'stress' of smoking or a pharmacologic action of nicotine and other tobacco substrates. Nicotine exposure is controlled in this study by allowing each subject to smoke only two 2.0 mg nicotine cigarettes during one experimental session and two 0.2 mg nicotine cigarettes in another session. Plasma levels of cortisol, growth hormone, and prolactin for the higher nicotine session were found to be significantly elevated over those for the low-nicotine session, indicating that nicotine itself plays a predominate role in smoking-induced hormone increases. All hormone levels for the 2.0 mg nicotine session had not returned to baseline 60 min after smoking.

Association of intrauterine cigarette smoke exposure with indices of fetal lung maturation.

The timing of fetal lung maturation is regulated, at least in part, by the fetal endocrine milieu, which in turn may be influenced by environmental factors. Infants of smoking mothers are at decreased risk of neonatal respiratory distress syndrome (RDS), a disease of lung immaturity. Therefore, we measured fetal lung maturity and cigarette smoke exposure to determine whether the lungs of smoke-exposed fetuses mature more quickly and whether changes in maturation are associated with alterations in amniotic fluid (AF) cortisol levels. Amniotic fluid lecithin-sphingomyelin ratio (L/S) and saturated phosphatidylcholine levels were used as measures of lung maturity, while smoke exposure was assessed by measuring AF cotinine, a stable nicotine metabolite. Lung maturity was more advanced in smoke-exposed fetuses as measured by saturated phosphatidylcholine (P = .02) and L/S ratio (P = .04). Smoke-exposed fetuses attained sufficient lung maturity to minimize the risk of RDS approximately 1 week earlier than in unexposed fetuses. The AF of smoke-exposed fetuses also had higher levels of free, conjugated, and total cortisol. Acceleration of lung maturation in smoke-exposed fetuses is consistent with the decreased risk of RDS in infants of smoking mothers. Maternal smoking could influence lung maturation by directly or indirectly enhancing the production and/or secretion of cortisol. Despite the decreased risk of RDS, the developmental process by which smoke-exposed fetuses attain early pulmonary maturity is abnormal and may contribute to the decreased lung function and increased respiratory illness noted in infants of smoking mothers.

The effects of smoking schedules on cessation outcome: can we improve on common methods of gradual and abrupt nicotine withdrawal?

This study compared the efficacy of 2 traditional methods of smoking cessation, gradual reduction and "cold turkey," with a new approach involving variation in the intercigarette interval. One hundred twenty-eight participants quit smoking on a target date, after a 3-week period of (a) scheduled reduced smoking (progressive increase in the intercigarette interval), (b) nonscheduled reduced smoking (gradual reduction, no specific change in the intercigarette interval), (c) scheduled nonreduced smoking (fixed intercigarette interval, no reductions in frequency), or (c) nonscheduled nonreduced smoking (no change in intercigarette interval or smoking frequency). Participants also received cognitive-behavioral relapse prevention training. Abstinence at 1 year averaged 44%, 18%, 32%, and 22% for the 4 groups, respectively. Overall, the scheduled reduced group performed the best and the nonscheduled reduced group performed the worst. Both scheduled groups performed better than nonscheduled ones. Scheduled reduced smoking was associated with reduced tension, fatigue, urges to smoke, withdrawal symptoms, increased coping effort (ratio of coping behavior to urges), and self-efficacy, suggesting an improved adaptation to nonsmoking and reduced vulnerability to relapse.

Behavior therapy and the transdermal nicotine patch: effects on cessation outcome, affect, and coping.

The process and outcome of a smoking cessation program using behavior therapy alone (BT) or behavior therapy plus the nicotine patch (BTP) was studied in 64 participants. Participants quit smoking on a target date after a period of ad libitum smoking, cognitive-behavior therapy preparing them for cessation, and behavioral rehearsal for high-risk situations, including stress management, and coping with negative affect. Abstinence was significantly higher for the BTP group versus the BT group from the end of behavioral treatment (79% vs. 63%) through the 3-month follow-up (p < .01), with the effects weakening at the 6- (p = .06) and 12-month marks (p = 38% vs. 22%). More general distress was observed among BT versus BTP participants (i.e., increased withdrawal, tension, fatigue, and coping frequency with decreased coping effort; coping-to-urge ratio). The coping behavior of the BTP group may have been more effective than that of the BT group, as indicated by their significantly higher level of self-efficacy.

How should urinary cotinine concentrations be adjusted for urinary creatinine concentration?

The correlation between cotinine concentrations in a single specimen of urine and in serum collected on the same occasion from 279 male smokers was 0.83. This was significantly increased, to 0.91, by adjusting the urinary cotinine levels for urinary creatinine concentration to take account of variations in urinary dilution between people. The adjustment used was based on the observed regression relationship between urinary cotinine and urinary creatinine concentrations. Expressing urinary cotinine values as a ratio to urinary creatinine, which has been used as a method of adjustment by others, did not improve the correlation between serum and urinary cotinine levels. The method of adjusting urinary cotinine for urinary creatinine is, therefore, important. The principle of such adjustment should apply not only to cotinine but also to other urinary biochemical measurements.

Production of antibodies to palytoxin: neutralization of several biological properties of palytoxin.

Palytoxin stimulated arachidonic acid metabolism (in bovine aorta endothelial and smooth muscle cells, rat keratinocytes, porcine aorta endothelial cells and rat liver cells), hemolyzed rat erythrocytes and was lethal to mice when administered intraperitoneally. Serum from rabbits immunized with a conjugate in which palytoxin was covalently bound to bovine albumin through its free amino group neutralized these biologic activities of palytoxin. Ninety-nine per cent of the neutralizing activity of the immunized rabbit serum was removed after precipitation of the rabbit IgG with a goat anti-rabbit IgG.

A radioimmunoassay for palytoxin.

Palytoxin, labelled with 125I-Bolton-Hunter reagent on its terminal amino group, bound specifically to rabbit anti-palytoxin. The extent of binding increased progressively with repeated immunizations. After absorption of the rabbit IgGs with a goat anti-rabbit IgG, binding was reduced greater than 95%. For 50% inhibition of binding in the 125I-palytoxin-antipalytoxin reaction 0.27 pmoles of unlabelled palytoxin was required. Maitotoxin, teleocidin, okadaic acid, debromoaplysiatoxin and 12-O-tetradecanoylphorbol-13-acetate, when tested at 10-100-fold higher concentrations than palytoxin did not affect binding. Palytoxin's serologic activity was stable after 60 min exposure to 100 degrees C and after 60 min exposure to 0.1 N HCl at 50 degrees C, but its capacity to stimulate the arachidonic acid metabolism of rat liver cells was reduced after the 60 min exposure to 0.1 N HCl treatments at 35 degrees C or 0.01 N HCl at 50 degrees C. The average binding constant (K0) as determined by separation of antibody-bound palytoxin from free palytoxin by the double antibody technique was 4.9 x 10(9) M-1 at 0 degrees C. This apparent average association constant increased with increasing temperature suggesting that palytoxin's epitope, most likely hydrophilic, is bound to H2O and the H2O is displaced before binding to the antibody's paratope.

Production of antibodies and development of a radioimmunoassay for okadaic acid.

An okadaic acid immunogen, prepared by conjugation of okadaic acid to bovine albumin with carbodiimide, was used to immunize two rabbits. The rabbits responded by producing antibodies that neutralized okadaic acid's stimulation of arachidonic acid metabolism and this neutralization increased during the course of immunization. The immune sera bound 3H-okadaic acid and this binding also increased with repeated immunization. After absorption of the rabbit IgG with a goat anti-rabbit IgG, binding was reduced greater than 99%. The binding of okadaic acid to the antibodies in one antiserum was inhibited by as little as 0.2 pmoles of unlabelled okadaic acid. The apparent association constant for binding with this antiserum was 4.17 x 10(9) M-1 (35 degrees C). Maitotoxin, teleocidin, 12-O-tetradecanoylphorbol-13-acetate, aplysiatoxin, palytoxin and brevetoxin B when tested at 29, 228, 168, 169, 3.7 and 112 pmole levels, respectively, did not inhibit binding. The serologic and biological activities of okadaic acid after incubation for 60 min in 0.01 N HCl at 35 degrees C or at 100 degrees C at pH 7.2 were unaffected.

The effects of smoking on the mood, cardiovascular and adrenergic reactivity of heavy and light smokers in a non-stressful environment.

Following a period of overnight deprivation, 58 smokers participated in a 90-min laboratory assessment in which they viewed a non-stressful movie and smoked two 0.5-mg nicotine-containing cigarettes. The first cigarette was given to all subjects following 25 min of adaptation and baseline. The next cigarette was provided at their request, which occurred 9-12 min later. "Heavy" and "light" smokers were grouped according to their average morning cotinine values, which fell above or below 250 ng/ml, respectively. The results showed that, relative to their baseline, heavy and light smokers experienced about the same level of post-smoking change in blood nicotine, heart rate and blood pressure. However, heavy smokers showed a significantly greater delta from baseline in post-smoking measures of epinephrine, norepinephrine, tension reduction and increase in vigor enhancement. A strong and consistent correlation was observed between post-smoking increases in epinephrine, tension reduction and increased vigor.

Nicotine regulation among heavy and light smokers in a non-stressful environment.

The purpose of this study was to assess nicotine regulation among "heavy" and "light" smokers. Previous studies supporting the nicotine regulation model of smoking behavior have suggested that smokers compensate for a reduction in the amount of nicotine available in their cigarette by altering smoking frequency, puff volume, or other aspects of smoking topography. However, little is known about a smoker's decision to smoke a specific cigarette, and the concurrent changes in their blood nicotine. Manipulation of nicotine levels in the blood could play a critical role in smoking maintenance, by regulating the extent and quality of the CNS effects of smoking. In this study, 24 heavy and light smokers (cotinine above or below 260 ng/ml) smoked high- (1.0 mg) or low- (0.5 mg) dose nicotine cigarettes while watching non-stressful movies. Blood nicotine was assessed before and after smoking a preload and free operant cigarette. The results showed that blood nicotine levels after smoking the free operant cigarette were significantly more consistent (lower standard error) for the heavy smokers, following a low dose, as opposed to a high-dose preload. Light smokers showed a non-significant trend towards being more consistent when the high-dose nicotine preload was used. This suggests that heavy smokers may have maximized their dose of nicotine whenever available nicotine was in relatively short supply (low dose condition). However, light smokers may have minimized their exposure when available nicotine was relatively more plentiful (high dose condition).

Decreased serum cotinine levels in smokers of both tobacco and marijuana as compared with smokers of tobacco only.

Serum and salivary cotinine levels were determined in tobacco smokers (n = 125) who smoked only tobacco (n = 47) or who smoked both marijuana and tobacco (n = 78) as part of a field study of the pulmonary effects of heavy, habitual use of marijuana alone or with tobacco. After adjustment for current daily amount of tobacco use and time since the last tobacco cigarette was smoked, the smokers of both marijuana and tobacco were found to have lower levels of cotinine than those who smoked only tobacco, in serum [258 +/- 113 ng/ml (S.D.) and 332 +/- 109, respectively; p = 0.003] and in saliva (331 +/- 170 and 395 +/- 170, respectively; p = 0.058). Serum cotinine showed a significantly negative relationship to the daily amount of marijuana currently smoked (p = 0.026). Possible explanations include inhibition by marijuana component(s) of the enzymes that participate in the conversion of nicotine to cotinine, differences in nicotine absorption patterns between the two groups of tobacco smokers, and acceleration of cotinine metabolism by marijuana smoking. Carefully controlled pharmacokinetic studies, not possible in a large-scale survey such as this one, are required both to confirm the differences in blood cotinine levels observed between the dual smokers and smokers of tobacco only and to define more clearly nicotine-marijuana interactions.

An evaluation of a multicomponent treatment program involving scheduled smoking and relapse prevention procedures: initial findings.

In the current study, 34 smokers were treated in a smoking cessation program that involved either a scheduled smoking procedure, or a minimal contact self-help treatment control. The interval smoking program consisted of baseline, cessation, and relapse prevention phases. During baseline, subjects self-monitored smoking and the total hours spent awake. During a 3-week cessation period, the scheduled smoking group progressively increased their intercigarette interval, thereby gradually reducing their total daily intake of nicotine. Smokers were expected to quit on a target date set at the end of this period. Cognitive behavioral interventions and relapse prevention training consisted of behavioral rehearsal of nonsmoking skills in a relapse prone environment. Control subjects were given the American Cancer Society "I Quit Kit", and provided subsequent discussion of its use. The results showed that 53% and 41% of the scheduled smoking group was abstinent at the 6- and 12-month follow-up points, respectively. Controls averaged only 6% for the same periods. Scheduled smoking may be a useful addition to a multicomponent treatment program and further study appears warranted to determine the saliency of the treatment features.