A Rare Case of Fibrous Hamartoma of Infancy: A Clinicopathological Diagnosis at a Tertiary Hospital, Eastern Nepal.

BACKGROUND: Fibrous hamartoma of infancy is a rare soft tissue lesion of infants and young children with characteristic triphasic morphology. CASE DESCRIPTION: An 18-month-old female child was presented with complaints of swelling over right leg shin since birth. On examination, a lump of size 7x3 cm was identified which was mobile and nontender. Local excision was performed and tissue sent for histopathological examination. On gross examination, a globular, capsulated, firm to hard tissue had cut section revealing solid grey-white to grey-brown lesion with myxoid areas identified. Microscopic examination revealed a poorly circumscribed lesion comprising intersecting trabeculae of fibrous tissue, areas of immature oval and stellate cell within myxoid matrix, and varying amounts of interspersed mature fat cells. CONCLUSION: Even though fibrous hamartoma of infancy is a rare benign entity with limited clinical knowledge, proper diagnosis is mandatory as its prognosis is excellent.

Muscular Hamartoma of Intestine Causing Intestinal Obstruction.

Hamartomatous causes of small bowel obstructionare uncommon and of them, most are attributed to inflammatory bowel diseases and also certain medications such as NSAIDs. We describe a case of muscular hamartoma in a patient without prior chronic medical condition with brief review of literature.

Lipopolysaccharide enhances mouse lung tumorigenesis: a model for inflammation-driven lung cancer.

The association between pulmonary inflammation and lung cancer is well established. However, currently there are no appropriate models that recapitulate inflammation-related lung cancer in humans. In the present study, we examined, in 2 tumor bioassays, enhancement by bacterial lipopolysaccharide (LPS) of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in A/J mice. Mice that were treated with NNK alone developed 29.6 ± 9.8 and 36.2 ± 4.1 lung tumors per mouse in experiments 1 and 2, respectively. Chronic intranasal instillation of LPS to NNK-treated mice increased the multiplicity of lung tumors to 47.3 ± 16.1 and 51.2 ± 4.8 lung tumors per mouse in experiments 1 and 2, corresponding to a significant increase by 60% and 41%, respectively. Moreover, administration of LPS to NNK-pretreated mice significantly increased the multiplicity of larger tumors and histopathologically more advanced lesions (adenoma with dysplasia and adenocarcinoma), macrophage recruitment to the peritumoral area, and expression of inflammation-, cell proliferation-, and survival-related proteins. Overall, our findings demonstrated the promise of the NNK-LPS-A/J mice model to better understand inflammation-driven lung cancer, dissect the molecular pathways involved, and identify more effective preventive and therapeutic agents against lung cancer.

Orbital rhabdomyosarcoma in an adult.

INTRODUCTION: Rhabdomyosarcoma is the most common primary orbital malignant tumor in children. Orbital lesions represent about 10 % of all the cases of rhabdomyosarcoma. Rhabdomyosarcoma is a rare cause of proptosis in adults. OBJECTIVE: To report a case of primary orbital rhabdomyosarcoma in a 45-year-old female. DESIGN: Interventional case report. The main outcome measures are a rare cause ofproptosis in an adult, discussion on treatment options and prognosis ofrhabdomyosarcoma. RESULT: The patient underwent total orbital exenteration and was referred for radiotherapy and chemotherapy. CONCLUSION: Rhabdomyosarcoma is a rare cause of proptosis in adults. It should be suspected in a case of rapidly-progressive proptosis in adults.

Abdominal wall endometriosis.

Endometriosis of abdominal wall scar following operation on uterus and tubes is extremely rare. The late onset of symptoms after surgery is the usual cause of misdiagnosis. Scar endometriosis is a rare disease which is difficult to diagnose and should always be considered as a differential diagnosis of painful abdominal masses in women. The diagnosis is made only after excision and histopathology of the lesion. Preoperative differentials include hernia, lipoma, suture granuloma or abscess. Hence an awareness of the entity avoids delay in diagnosis, helps clinicians to a more tailored treatment and also avoids unnecessary referrals. We report a case of abdominal endometriosis. The definitive diagnosis of which was established by histopathological studies.

Comparative study for the presence of enterococcal virulence factors gelatinase, hemolysin and biofilm among clinical and commensal isolates of enterococcus faecalis.

BACKGROUND: Biofilm production, gelatinase and hemolysin are the potential virulence factors of Enterococci. Gelatinase and hemolysin producing strains of Enterococcus Faecalis have been shown to cause severe infections in animal models. Biofilm production has been shown to enhance the persistence of E. faecalis in urinary bladder and other medical indwelling devices infections. AIMS: To compare the presence of gelatinase, hemolysin and biofilm formation among clinical and commensal isolates and to study the co-relation between virulence factors with respect to different clinical specimens. SETTINGS AND DESIGN: During the study period of 2 years from July 2004 to July 2006, 200 clinical isolates from nosocomial infections and 100 commensal isolates of E. faecalis were taken for the study. MATERIALS AND METHODS: The clinical and commensal isolates were tested for the presence of gelatinase, hemolysin and biofilm and compared. The presence of these virulence factors among different clinical isolates was also studied. STATISTICAL ANALYSIS: Chi-square and likelihood ratio analysis were carried out using SSPS version 5.1 software. RESULTS: The clinical isolates produced 39, 16.5 and 32.5% of gelatinase, hemolysin and biofilm, respectively, as compared to 31, 19 and 16% produced by the commensal isolates, respectively. Endotracheal tube infection, urinary tract infection, umbilical catheter tip infected isolates produced 60.8, 86.6 and 100% biofilm, respectively. CONCLUSION: Significant difference in the production of biofilm (P<0.001) was noted between clinical and commensal isolates. Organism isolated from medically indwelling devices produced high amount of biofilm, confirming its role in colonization and causing nosocomial infections.

Review of virulence factors of enterococcus: an emerging nosocomial pathogen.

Enterococcus, considered a normal commensal of intestinal tract, is fast emerging as a pathogen causing serious and life threatening hospital borne infections. This is attributed to acquisition of multi drug resistance and virulence factors of the organisms. The sequencing of Enterococcus faecalis has given a lot of insight into its genetic makeup. The E. faecalis strain V583, which has been sequenced, contains a total of 3182 open reading frames (ORFs) with 1760 of these showing similarity to known proteins and 221 of unknown functions. Strikingly unique to this genome is the fact that over 25% of the genome is made up of mobile and exogenously acquired DNA which includes a number of conjugative and composite transposons, a pathogenicity island, integrated plasmid genes and phage regions, and a high number of insertion sequence (IS) elements. This review addresses the genomic arrangement and the study of virulence factors that have occurred since the sequencing of the genome.

Antiproliferative effect of 1,4-phenylenebis(methylene)selenocyanate (p-XSC) on colonic epithelium of patients with adenomatous polyps in vitro.

We have consistently shown that the organoselenium compound 1,4-phenylenebis(methylene)selenocyanate (p-XSC) is a superior cancer chemopreventive agent and less toxic than selenite or certain naturally-occurring selenoamino acids. To elucidate the effects of p-XSC on human colonic mucosa, biopsies from endoscopically normal sigmoid colon of 30 patients with adenomatous polyps were incubated with p-XSC at concentrations of 1, 2 and 5 micromol/l dissolved in dimethylsulphoxide (DMSO). Biopsies incubated with DMSO or pure culture medium served as a control. Proliferating cells were labelled by bromodeoxyuridine immunohistochemistry and the labelling index (LI) was computed. Upper crypt labelling index (LI of crypt compartments 4+5) and Phih value, which are both discriminators of the expansion of the proliferative zone, were significantly lower after incubation with 1 and 5 micromol/l p-XSC, respectively (LI 4+5: 0.8 and 1.0; Phih value: 2.1 and 2.4), as compared with DMSO (LI 4+5: 3.6 and 4.5; Phih value: 7.0 and 8.3) or culture medium (LI 4+5: 3.3 and 4.5; Phih value: 7.2 and 8.1) (P<0.005 and P<0.05 by Friedman's block test). A trend towards lower levels of LI 4+5 (P=0.059) and Phih value (P=0.075) were seen after 2 micromol/l p-XSC incubation compared with DMSO. Since hyperproliferation of colonic crypt cells with expansion of the proliferative zone is regarded as a biomarker of increased cancer risk, the antiproliferative effects of p-XSC especially on upper crypt LI and Phih value may indicate a possible protective effect of this organoselenium compound in the prevention of human colon cancer development.

Conservative management of splenic trauma: history and current trends.

Evolution of the present-day policy of conservative management of ruptured spleen has been hailed as one of the most notable advances in pediatric surgery. Until 1971, routine splenectomy used to be the sacrosanct treatment for splenic trauma. It was universally believed that non-operative management carried a high mortality of 90 to 100%. Sporadic reports of successful conservative treatment appeared in the early twentieth century, but regrettably, these were ignored. Likewise, experimental studies pointing to the danger of post-splenectomy sepsis were also disregarded. Dominant surgical opinion continued to practice removal of the injured spleen. In 1968, Upadhyaya and Simpson, based on a well-designed clinical analysis of 52 children made a convincing plea for conservative management. In 1971, Upadhyaya et al. presented results of a corroborative experimental study, which provided the conclusive evidence that isolated splenic tears are well tolerated and heal spontaneously by first intention. Seeing the surge of publications that followed this presentation, it becomes apparent that this study constituted the real turning point that changed the world opinion in favour of salvage of the ruptured spleen. By 1979, numerous authors had reported the safety of non-operative management in hundreds of children all over the world. Currently, the policy of routine splenectomy has been universally abandoned; and the reported salvage rate of ruptured spleen is more than 90%. This paper traces the historical perspectives in the management of injured spleen from the times of Aristotle to the present day.

Reactions of alpha-acetoxy-N-nitrosopyrrolidine with deoxyguanosine and DNA.

We investigated the reactions of alpha-acetoxy-N-nitrosopyrrolidine (alpha-acetoxyNPYR) with dGuo and DNA. Alpha-acetoxyNPYR is a stable precursor to the major proximate carcinogen of NPYR, alpha-hydroxyNPYR (3). Our goal was to develop appropriate conditions for the analysis of DNA adducts of NPYR formed in vivo. Products of the alpha-acetoxyNPYR-dGuo reactions were analyzed directly by HPLC or after treatment of the reaction mixtures with NaBH3CN. Products of the alpha-acetoxyNPYR-DNA reactions were released by enzymatic or neutral thermal hydrolysis of the DNA, then analyzed by HPLC. Alternatively, the DNA was treated with NaBH3CN prior to hydrolysis and HPLC analysis. The reactions of alpha-acetoxyNPYR with dGuo and DNA were complex. We have identified 13 products of the dGuo reaction-6 of these were characterized in this reaction for the first time. They were four diastereomers of N2-(3-hydroxybutylidene)dGuo (20, 21), 7-(N-nitrosopyrrolidin-2-yl)Gua (2), and 2-(2-hydroxypyrrolidin-1-yl)deoxyinosine (12). Adducts 20 and 21 were identified by comparison to standards produced in the reaction of 3-hydroxybutanal with dGuo. Adduct 2 was identified by its spectral properties while adduct 12 was characterized by comparison to an independently synthesized standard. With the exception of adduct 2, all products of the dGuo reactions were also observed in the DNA reactions. The major product in both the dGuo and DNA reactions was N2-(tetrahydrofuran-2-yl)dGuo (10), consistent with previous studies. Several other previously identified adducts were also observed in this study. HPLC analysis of reaction mixtures treated with NaBH3CN provided improved conditions for adduct identification, which should be useful for in vivo studies of DNA adduct formation by NPYR.

Inhibition of vascular endothelial cells by 1,4-phenylenebis (methylene)selenocyanate--a novel chemopreventive organoselenium compound.

Organoselenium compound 1,4-phenylenebis(methylene)selenocyanate (p-XSC) was investigated for its effects on endothelial cell proliferation in vitro and angiogenesis in vivo. The organoselenium compound, p-XSC, has been shown to prevent carcinogen-induced tumorigenesis in murine model systems with low toxicity. Since tumor growth and metastasis are dependent on angiogenesis, we investigated the effects of the organoselenium compound on this process. Human umbilical vein endothelial cells treated with p-XSC showed concentration dependent inhibition of protein synthesis and cell viability in vitro with a TCID50 value of 6 microM. Subsequently, we studied the effects of p-XSC on experimental angiogenesis. Addition of p-XSC to three-dimensional cultures inhibited endothelial cell tube formation. Furthermore, p-XSC treatment inhibited growth factor induced angiogenesis in chick chorioallantoic membrane assays and i.p. administration of p-XSC inhibited neovascularization induced by tumor cells implanted subcutaneously into athymic mice. These studies suggest that vascular endothelium is an additional target for the chemopreventive organoselenium compound p-XSC.

Preparation of pyridine-N-glucuronides of tobacco-specific nitrosamines.

Nicotine and cotinine are metabolized to pyridine-N-glucuronides in humans. This suggests that the analogous metabolites of the carcinogenic nicotine-related nitrosamines N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) should also be formed in people exposed to these compounds via tobacco products. We describe the synthesis of the appropriate pyridine-N-glucuronides: pyridyl-N-beta-D-glucopyranuronosyl-N'-nitrosonornicotinium inner salt (NNN-N-Gluc, 8), 4-(methylnitrosamino)-1-(3-pyridyl-N-beta-D-glucopyranuronosyl)-1-butanonium inner salt (NNK-N-Gluc, 9), and 4-(methylnitrosamino)-1-(3-pyridyl-N-beta-D-glucopyranuronosyl)-1-butanolonium inner salt (NNAL-N-Gluc, 10). The starting material, methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-alpha-D-glucopyranuronate (1), is prepared in two steps from glucuronolactone. Reactions of 1 with racemic NNN (2), NNK (3), or racemic NNAL (4) are carried out with no solvent and the crude products are deprotected by treatment with base, giving the desired N-glucuronides 8-10 in 5-7% overall yield after HPLC purification. The N-glucuronides were characterized by (1)H NMR, including COSY and NOESY spectra, and by MS and MS/MS. NNN-N-Gluc exists as a 52:48 ratio of (E)- and (Z)-rotamers, which were partially separated by HPLC. This ratio was surprisingly similar to the (E):(Z) ratio for NNN itself suggesting hydrogen bonding of the (Z)-nitroso oxygen atom to the 2' '-hydroxyl group of the glucuronide moiety. Partial HPLC separations of the (E)- and (Z)-rotamers of NNK-N-Gluc and the (E)- and (Z)-rotamers as well as the (R)- and (S)-diastereomers of NNAL-N-Gluc were also achieved. The standards prepared in this study as well as the HPLC conditions developed for their separation will be important for analysis of these compounds in human urine.

Transport of the beta -O-glucuronide conjugate of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) by the multidrug resistance protein 1 (MRP1). Requirement for glutathione or a non-sulfur-containing analog.

Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) play a crucial role in the induction of lung cancer, and NNAL-O-glucuronide formation and elimination are important steps in detoxification of these compounds. In the present study, we investigated the ATP-binding cassette (ABC) protein, MRP1 (ABCC1), as a candidate transporter responsible for NNAL-O-glucuronide export. MRP1 mediates the active transport of numerous GSH-, sulfate-, and glucuronide-conjugated organic anions and can transport certain xenobiotics by a mechanism that may involve co-transport with GSH. Using membrane vesicles prepared from transfected cells, we found that MRP1 transports [3H]NNAL-O-glucuronide but is dependent on the presence of GSH (Km 39 microm, Vmax 48 pmol x mg(-1) x min(-1)). We also found that the sulfur atom in GSH was dispensable because transport was supported by the GSH analog, gamma-glutamyl-alpha-aminobutyryl-glycine. Despite stimulation of NNAL-O-glucuronide transport by GSH, there was no detectable reciprocal stimulation of [3H]GSH transport. Moreover, whereas the MRP1 substrates leukotriene C4 (LTC4) and 17beta-estradiol 17beta-(d-glucuronide) (E(2)17betaG) inhibited GSH-dependent uptake of [3H]NNAL-O-glucuronide, only [3H]LTC4 transport was inhibited by NNAL-O-glucuronide (+GSH) and the kinetics of inhibition were complex. A mutant form of MRP1, which transports LTC4 but not E(2)17betaG, also did not transport NNAL-O-glucuronide suggesting a commonality in the binding elements for these two glucuronidated substrates, despite their lack of reciprocal transport inhibition. Finally, the related MRP2 transported NNAL-O-glucuronide with higher efficiency than MRP1 and unexpectedly, GSH inhibited rather than stimulated uptake. These studies provide further insight into the complex interactions of the MRP-related proteins with GSH and their conjugated organic anion substrates, and extend the range of xenotoxins transported by MRP1 and MRP2 to include metabolites of known carcinogens involved in the etiology of lung and other cancers.

Dose-response study of myo-inositol as an inhibitor of lung tumorigenesis induced in A/J mice by benzo.

Dietary myo-inositol is an effective inhibitor of lung tumor induction in mice, but no dose-response studies have been reported. We assessed the ability of various doses of dietary myo-inositol to inhibit lung tumor induction in female A/J mice treated with eight weekly doses of benzo[a]pyrene (BaP) plus 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (3 micromol of each by gavage), then killed 18 weeks later. In Expt. 1, groups of 20 mice each were treated with myo-inositol at concentrations of 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, and 0% in AIN-93 diet for 1 week prior to, during, and for 1 week after the carcinogen administration period. In Expt. 2, groups of 20 mice each were treated with the same concentrations of myo-inositol in the diet as in Expt. 1, except this diet was administered from 1 week after carcinogen administration until termination. There were no effects of myo-inositol on lung tumor incidence, which was 100% in all groups treated with BaP plus NNK. However, myo-inositol significantly decreased lung tumor multiplicity in both experiments. In Expt. 1, significant reductions of 28.9 and 33.0% were observed at the 1 and 0.5% doses of myo-inositol, but not at the lower doses. In Expt. 2, a significant reduction of 48.4% was observed at the 1% dose. In both Expts. 1 and 2, there was a significant dose trend for inhibition (P<0.0001). No toxicity was observed at any dose. These results firmly establish myo-inositol as a chemopreventive agent against lung tumor induction in A/J mice, at doses that can be envisioned for human use.

Formation and metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol enantiomers in vitro in mouse, rat and human tissues.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is a major metabolite of the tobacco-specific lung carcino- gen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). NNAL has a chiral center at the 1-position, but little is known about the stereochemical aspects of its metabolic formation from NNK or its further metabolism. We investigated the metabolism of NNK to enantiomers of NNAL in microsomes and cytosol from male F-344 rat liver and lung, female A/J mouse liver and lung, and human liver, as well as in red blood cells from rats, mice and humans. In all systems, (S)-NNAL was the predominant enantiomer formed, ranging from 90 to 98% in the rodent tissues and averaging 64, 90 and >95% in human liver microsomes, liver cytosol and red blood cells, respectively. In rat liver microsomes, (R)- and (S)-NNAL were metabolized at similar rates by alpha-hydroxylation, considered to be the major metabolic activation pathway of NNAL. Pyridine-N-oxidation and adenosine dinucleotide phosphate adduct formation also occurred at similar rates from both enantiomers, while reoxidation to NNK was favored with (S)-NNAL as substrate. In rat lung microsomes, (S)-NNAL was more rapidly metabolized than (R)-NNAL by all oxidative pathways. In human liver microsomes, there were no significant differences in the rates of alpha-hydroxylation, pyridine-N-oxidation and reoxidation to NNK between the two enantiomers. The results of this study demonstrate that (S)-NNAL, the more tumorigenic enantiomer in mice, is preferentially formed from NNK in rodent and human tissues, and is a substrate for oxidative metabolism in rodent and human tissue microsomes.

Mechanisms in the chemoprevention of colon cancer: modulation of protein kinase C, tyrosine protein kinase and diacylglycerol kinase activities by 1,4-phenylenebis-(methylene)selenocyanate and impact of low-fat diet.

Epidemiological and experimental studies suggest an inverse relationship between the intake of dietary selenium and/or low fat-intake and colon cancer risk. Efficacy studies in rodents suggest that the organoselenium compound 1, 4-phenylenebis(methylene)selenocyanate (p-XSC), is a more effective and less toxic chemopreventive agent than other organic or inorganic selenium compounds such as selenomethionine and Na2SeO3. The efficacy of p-XSC against colon cancer is significantly augmented by a low-fat diet. To explore the mechanisms by which this combined inhibiting effect against colon carcinogenesis comes about, we have investigated protein kinase C (PKC), tyrosine protein kinase (TPK), diacylglycerol kinase (DGK) activities and 8-isoprostane levels in colonic mucosa and tumor tissues in an azoxymethane (AOM)-induced rat colon cancer model. Weanling male F344 rats were fed the semipurified AIN-76A diet until seven weeks of age. Then various experimental groups were fed the low- or high-fat diets containing 0 or 20 ppm p-XSC (10 ppm as selenium). At seven weeks of age, groups of rats were injected s.c. with azoxymethane (AOM; 15 mg/kg body wt., once weekly for 2 weeks) and continued on their respective experimental diets until 38 weeks after the second AOM treatment. They were then sacrificed and colonic mucosal and tumor samples were evaluated for PKC, TPK, DGK and 8-isoprostane levels. Administration of p-XSC along with a low-fat diet significantly inhibited Ca+2-dependent and -independent PKC (P<0.05-0.01) activities in colonic mucosa and tumors. Administration of p-XSC either low-fat or high-fat diet significantly suppressed both colonic mucosal and tumor TPK activity (P<0.05-0.01). Suppression of TPK activity was more pronounced in rats maintained on a low-fat diet containing p-XSC. In contrast, rats receiving p-XSC with either low- or high fat diet showed significantly increased DGK activity (P<0.01-0.0001). Rats fed low-fat or high-fat plus p-XSC had lower-levels of 8-isoprostane in the colonic tumors than animals who had been given low- or high-fat diets without the organoselenium compound. Interestingly, 8-isoprostane levels were lower in the colon tumors of the rats fed the low-fat diet than those fed the high-fat diet. Our findings suggest that p-XSC induced down-regulation of PKC and TPK activities and up-regulation of DGK activity. These events may in part be responsible for the chemopreventive activity against colon carcinogenesis. Further, this study implies that p-XSC with a low-fat dietary regimen will augment regulation of PKC, TPK and DGK activities in the colon.

Effects of phenethyl isothiocyanate and benzyl isothiocyanate, individually and in combination, on lung tumorigenesis induced in A/J mice by benzo[a]pyrene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

Phenethyl isothiocyanate (PEITC) is an effective inhibitor of lung tumorigenesis induced in rats and mice by the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) while benzyl isothiocyanate (BITC) inhibits lung tumorigenesis induced in mice by another tobacco smoke carcinogen, benzo[a]pyrene (BaP). However, little is known about the inhibitory effects of PEITC and BITC in combination, or about the effects of PEITC or BITC on tumorigenesis by a mixture of NNK and BaP. In this study, we carried out a series of experiments pertinent to these questions. In Experiment 1, treatment of A/J mice with PEITC (6 micromol), BITC (6 micromol), or a combination of the two (6 micromol each) by gavage, 2 h prior to each of eight weekly gavage treatments with a mixture of BaP and NNK (3 micromol of each), had no effect on lung tumor multiplicity. In Experiment 2, we evaluated the inhibitory potential of four different mixtures of PEITC and BITC, administered by gavage 2 h prior to each of eight weekly doses of BaP and NNK, as given in Experiment 1. Mixtures of PEITC and BITC (12 micromol of each, or 12 micromol PEITC and 9 micromol BITC) significantly reduced lung tumorigenesis induced by a mixture of BaP and NNK. In Experiment 3, we investigated the effects of dietary PEITC (3 micromol/g diet), BITC (1 micromol/g diet), or a mixture of PEITC (3 micromol/g diet) and BITC (1 micromol/g diet). These compounds were started 1 week before, and continued through to 1 week after the eight weekly treatments with BaP and NNK. PEITC, and PEITC plus BITC, both significantly inhibited lung tumor multiplicity; inhibition was due mainly to PEITC. In Experiment 4, we tested dietary PEITC (3, 1, or 0.3 micromol/g diet) as an inhibitor of lung tumorigenesis induced by BaP, NNK, or BaP plus NNK using a protocol identical to that in Experiment 3. PEITC was an effective inhibitor of lung tumor multiplicity induced by NNK and a mixture of BaP plus NNK, but not by BaP. Dietary PEITC, or PEITC plus BITC, was more effective in these experiments than the compounds given by gavage. The results of this study demonstrate that proper doses of dietary PEITC and dietary as well as gavaged PEITC plus BITC are effective inhibitors of lung tumorigenesis induced in A/J mice by a mixture of BaP and NNK.

Stereochemistry of metabolites of a tobacco-specific lung carcinogen in smokers' urine.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific lung carcinogen, is believed to be important as a causative agent for lung cancer in smokers. NNK is extensively metabolized to its carbonyl reduction product 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which, in turn, can be glucuronidated, producing [4-methylnitrosamino)-1-(3-pyridyl)but-1-yl]-beta-O-D-glucosiduronic+ ++ acid (NNAL-Gluc). Metabolism of NNK to NNAL produces a chiral center. A recent study demonstrated that (R)-NNAL is more tumorigenic in mice than (S)-NNAL and that these enantiomers have substantially different metabolic pathways. Therefore, it is important to determine the stereochemistry of NNAL and NNAL-Gluc in smokers. In this study, we used chiral stationary phase-gas chromatography-nitrosamine-selective detection with confirmation by liquid chromatography-tandem mass spectrometry to determine the stereochemistry of NNAL and NNAL-Gluc in smokers' urine. The two methods agreed well. The results of analyses of urine samples from 30 smokers demonstrated that the enantiomeric distribution of NNAL in urine was 54% (R) and 46% (S) +/- 7.0 (SD), whereas the diastereomeric distribution of NNAL-Gluc was 68% (R) and 32% (S) +/- 8.1. These results conclusively demonstrate that both (R)- and (S)-NNAL are formed metabolically from NNK in smokers. These data are essential for furthering our understanding of the role of NNK as a cause of lung cancer in smokers.

Tumorigenicity and metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol enantiomers and metabolites in the A/J mouse.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a major metabolite of the tobacco-specific pulmonary carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), has a chiral center but the tumorigenicity of the NNAL enantiomers has not been previously examined. In this study, we assessed the relative tumorigenic activities in the A/J mouse of NNK, racemic NNAL, (R)-NNAL, (S)-NNAL and several NNAL metabolites, including [4-(methylnitrosamino)-1-(3-pyridyl)but-(S)-1-yl] beta-O-D-gluco-siduronic acid [(S)-NNAL-Gluc], 4-(methylnitrosamino)-1-(3-pyridyl N-oxide)-1-butanol, 5-(3-pyridyl)-2-hydroxytetrahydrofuran, 4-(3-pyridyl)butane-1,4-diol and 2-(3-pyridyl) tetrahydrofuran. We also quantified urinary metabolites of racemic NNAL and its enantiomers and investigated their metabolism with A/J mouse liver and lung microsomes. Groups of female A/J mice were given a single i.p. injection of 20 micromol of each compound and killed 16 weeks later. Based on lung tumor multiplicity, (R)-NNAL (25.6 +/- 7.5 lung tumors/mouse) was as tumorigenic as NNK (25.3 +/- 9.8) and significantly more tumorigenic than racemic NNAL (12.1 +/- 5.6) or (S)-NNAL (8.2 +/- 3.3) (P < 0. 0001). None of the NNAL metabolites was tumorigenic. The major urinary metabolites of racemic NNAL and the NNAL enantiomers were 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid), NNAL-N-oxide and NNAL-Gluc, in addition to unchanged NNAL. Treatment with (R)-NNAL or (S)-NNAL gave predominantly (R)-hydroxy acid or (S)-hydroxy acid, respectively, as urinary metabolites. While treatment of mice with racemic or (S)-NNAL resulted in urinary excretion of (S)-NNAL-Gluc, treatment with (R)-NNAL gave both (R)-NNAL-Gluc and (S)-NNAL-Gluc in urine, apparently through the metabolic intermediacy of NNK. (S)-NNAL appeared to be a better substrate for glucuronidation than (R)-NNAL in the A/J mouse. Mouse liver and lung microsomes converted NNAL to products of alpha-hydroxylation, to NNAL-N-oxide, to adenosine dinucleotide phosphate adducts and to NNK. In lung microsomes, metabolic activation by alpha-hydroxylation of (R)-NNAL was significantly greater than that of (S)-NNAL. The results of this study provide a metabolic basis for the higher tumorigenicity of (R)-NNAL than (S)-NNAL in A/J mouse lung, namely preferential metabolic activation of (R)-NNAL in lung and preferential glucuronidation of (S)-NNAL.

Evaluation of butylated hydroxyanisole, myo-inositol, curcumin, esculetin, resveratrol and lycopene as inhibitors of benzo[a]pyrene plus 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis in A/J mice.

The potential activities of butylated hydroxyanisole (BHA), myo-inositol, curcumin, esculetin, resveratrol and lycopene-enriched tomato oleoresin (LTO) as chemopreventive agents against lung tumor induction in A/J mice by the tobacco smoke carcinogens benzo[a]pyrene (BaP) and 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were evaluated. Groups of 20 A/J mice were treated weekly by gavage with a mixture of BaP and NNK (3 micromol each) for 8 weeks, then sacrificed 26 weeks after the first carcinogen treatment. Mice treated with BHA (20 or 40 micromol) by gavage 2 h before each dose of BaP and NNK had significantly reduced lung tumor multiplicity. Treatment with BHA (20 or 40 micromol) by gavage weekly or with dietary BHA (2000 ppm), curcumin (2000 ppm) or resveratrol (500 ppm) from 1 week after carcinogen treatment until termination had no effect on lung tumor multiplicity. Treatment with dietary myo-inositol (30,000 ppm) or esculetin (2000 ppm) from 1 week after carcinogen treatment until termination significantly reduced lung tumor multiplicity, with the effect of myo-inositol being significantly greater than that of esculetin. Treatment with dietary LTO (167, 1667 or 8333 ppm) from 1 week before carcinogen treatment until termination had no effect on lung tumor multiplicity. The results of this study demonstrate that BHA is an effective inhibitor of BaP plus NNK-induced lung tumorigenesis in A/J mice when administered during the period of carcinogen treatment and that, among the compounds tested, myo-inositol is most effective after carcinogen treatment.