Evidence that a hemoglobin adduct of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone is a 4-(3-pyridyl)-4-oxobutyl carboxylic acid ester.

Hemoglobin adducts of the carcinogenic tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) release 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) upon mild base or acid hydrolysis. HPB has been detected in hydrolysates of human hemoglobin and has been proposed as a dosimeter of exposure to and metabolic activation of NNK in people exposed to tobacco products. In this study, labeling experiments were carried out with Na18OH which provide strong evidence that the globin adduct which releases HPB upon base hydrolysis is a carboxylic acid ester. Globin was isolated from rats treated with NNK. This globin was reacted with NaCNBH3, followed by hydrolysis at room temperature with 0.2 N NaOH. Analysis of the products demonstrated the presence of 4-hydroxy-1-(3-pyridyl)-1-butanol (7), but not HPB. These results demonstrate that the adduct in globin has a free carbonyl group and is not a Schiff base. This sequence of reactions was then carried out with Na18OH, under conditions which would have resulted in incorporation of 18O into 7 if nucleophilic displacement at carbon 4 of the adduct had occurred. Analysis of the products by GC-MS showed no detectable incorporation of 18O into 7. These results demonstrate that the globin adduct which releases HPB upon base hydrolysis is a 4-(3-pyridyl)-4-oxobutyl carboxylic ester. Consistent with this conclusion, a model ester, alpha-methyl beta-[4-(3-pyridyl)-4-oxobutyl] N-(carbobenzyloxy)-L-aspartate (13), hydrolyzed in base and acid in a manner similar to that observed with globin from NNK-treated rats.     

Antagonism by propranolol of isolation-induced aggression in mice: Correlation with 5-hydroxytryptamine receptor blockade

The effects of β-adrenoceptor antagonists, (±)-propranolol, pindolol, metroprolol, atenolol and (+)-propranolol, and of 5-HT receptor antagonists, methysergide and cyproheptidine, were studied on the aggressive behaviour induced in male mice by prolonged isolation. A close correlation was found between the dose of drug required to antagonize 5-HTP-induced head twitches in mice and that which prevented fighting in previously isolated mice. Metroprolol, atenolol and (+) -propranolol were inactive in both tests, while the other agents all reduced aggressive behaviour in doses of 0.5–4 mg/kg. In a dose of 10 mg/kg, 5-HTP increased aggressive behaviour in isolated mice. It is suggested that fighting behaviour is associated with an increased activation of 5-HTP pathways in the C.N.S. β-Adrenoceptor antagonists prevent this behaviour because of their blockade of 5-HT receptors rather than those of nor-adrenaline.     

Immaturity of IL-18 gene expression and protein production in cord blood (CB) versus peripheral blood (PB) mononuclear cells and differential effects in natural killer (NK) cell development and function

We have previously demonstrated dysregulation of IL-12 and IL-15 gene and protein expression between activated cord blood (CB) versus peripheral blood (PB) mononuclear cells (MNCs). In the present study, we compared IL-18 gene expression and protein production and IL-18 mRNA half-life in basal versus activated CB versus PB MNCs, the effects of IL-18 +/- IL-12 on MNCs IFN-gamma protein production and ex vivo expansion and activation of CB with IL-12 + IL-2 + anti-CD3 +/- IL-18. Basal and activated levels of IL-18 were significantly higher in PB versus CB MNCs (P < 0.05). IL-18 mRNA was coincidental with protein levels and significantly lower in CB (P < 0.05) and its half-life significantly shorter in CB versus PB MNCs (P < 0.05). IL-18 synergistically with IL-12 induced IFN-gamma production from PB greater than CB MNCs (P < 0.05). NK cells expansion (P < 0.001) and cytotoxicity (P < 0.01) was significantly increased with IL-12 + IL-2 + anti-CD3 and IL-18. In summary IL-18 gene expression and protein production are significantly decreased in activated CB versus PB MNCs, in part secondary to increased degradation of CB IL-18 mRNA. These results may have implications for the mechanism(s) in part responsible for the immaturity of CB T-cell immunity.     

Quantitation of acrolein-derived (3-hydroxypropyl)mercapturic acid in human urine by liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry: effects of cigarette smoking

Recently published data suggest that acrolein (1), a toxic but weakly carcinogenic constituent of cigarette smoke, may be involved as a causative factor for the mutations frequently observed in the p53 tumor suppressor gene in lung cancer in smokers. Biomarkers are needed to further assess the possible relationship between acrolein uptake and cancer. In this study, we analyzed (3-hydroxypropyl)mercapturic acid (3-HPMA, 2) in human urine. 3-HPMA is a major metabolite of acrolein in laboratory animals. The method employs [13C3]3-HPMA as an internal standard, with analysis and quantitation by LC-APCI-MS/MS-SRM. Clean, readily quantifiable chromatograms were obtained. The method was accurate and precise and required only 0.1 mL of urine. Median levels of 3-HPMA were significantly higher (2900 pmol/mg of creatinine, N=35) in smokers than in nonsmokers (683 pmol/mg of creatinine, N=21) (P=0.0002). The effect of smoking was further assessed by determining the levels of 3-HPMA before and after a 4 week smoking cessation period. There was a significant 78% decrease in median levels of urinary 3-HPMA after cessation (P<0.0001). The relationship between the levels of urinary 3-HPMA and those of acrolein-derived 1,N2-propanodeoxyguanosine (PdG) adducts in lung was investigated in 14 smokers. There was a significant inverse relationship between urinary 3-HPMA and alpha-hydroxy-PdG (3) but not gamma-hydroxy-PdG (4) or total adduct levels. The results of this study clearly demonstrate that acrolein uptake in smokers is significantly higher than in nonsmokers and underline the need for further investigation of the possible relationship of acrolein uptake to lung cancer.     

Genetic variability in the metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL)

Urinary metabolites of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronides, termed total NNAL, have recently been shown to be good predictors of lung cancer risk, years before diagnosis. We sought to determine the contribution of several genetic polymorphisms to total NNAL output and inter-individual variability. The study subjects were derived from the Harvard/Massachusetts General Hospital Lung cancer case-control study. We analyzed 87 self-described smokers (35 lung cancer cases and 52 controls), with urine samples collected at time of diagnosis (1992-1996). We tested 82 tagging SNPs in 16 genes related to the metabolism of NNK to total NNAL. Using weighted case status least squares regression, we tested for the association of each SNP with square-root (sqrt) transformed total NNAL (pmol per mg creatinine), controlling for age, sex, sqrt packyears and sqrt nicotine (ng per mg creatinine). After a sqrt transformation, nicotine significantly predicted a 0.018 (0.014, 0.023) pmol/mg creatinine unit increase in total NNAL for every ng/mg creatinine increase in nicotine at p < 10E-16. Three HSD11B1 SNPs and AKR1C4 rs7083869 were significantly associated with decreasing total NNAL levels: HSD11B1 rs2235543 (p = 4.84E-08) and rs3753519 (p = 0.0017) passed multiple testing adjustment at FDR q = 1.13E-05 and 0.07 respectively, AKR1C4 rs7083869 (p = 0.019) did not, FDR q = 0.51. HSD11B1 and AKR1C4 enzymes are carbonyl reductases directly involved in the single step reduction of NNK to NNAL. The HSD11B1 SNPs may be correlated with the functional variant rs13306401 and the AKR1C4 SNP is correlated with the enzyme activity reducing variant rs17134592, L311V.