Treatment of renal artery aneurysms.

Renal artery aneurysms (RAAs) represent a rare disease and are most commonly discovered as an incidental imaging finding. RAA may be associated with hypertension and are usually asymptomatic at presentation but may result in rupture, hematuria, or renal infarction. The natural history of RAA is poorly understood. Although there is general consensus that RAA that are symptomatic or identified in women at risk for pregnancy should be repaired, diameter criteria for repair of asymptomatic RAA are controversial and emerging evidence suggests that rupture incidence is low for those <2.5 cm in diameter. Options for repair of RAA have expanded over the preceding decades with expansion of both open surgical and endovascular treatments.

Proxy measures of vitamin D status - season and latitude - correlate with adverse outcomes after bariatric surgery in the Nationwide Inpatient Sample, 2001-2010: a retrospective cohort study.

OBJECTIVE: To investigate the association between adverse surgical outcomes following bariatric surgery and proxy measures of vitamin D (VitD) status (season and latitude) in the Nationwide Inpatient Sample (NIS). BACKGROUND: Obesity is an independent risk factor for VitD deficiency (25(OH)D < 20 ng ml-1). VitD deficiency compounds the chronic inflammation of obesity, increasing the risk of adverse outcomes following bariatric surgery. Epidemiology has long used season and latitude as proxies for group VitD, as VitD status is largely determined by sun exposure, which is greatest during summer and at the Equator. METHODS: We assessed proxy measures of group VitD status. We compared surgeries in VitD Summer (July to September), Winter (January to March), and Fall/Spring (October to December and April to June) and in the North (≥37°N) vs. the South (<37°N). RESULTS: We identified 932,091 bariatric surgeries; 81.2% were women and 74.4% were white. Sex was unequally distributed by season (p = 0.005). Median age was 43.0 years (all groups). Most surgeries occurred in the North (64.8%). Adverse outcome rates ranged from 0.01% (wound infections) to 39.4% [prolonged length of stay {LOS}]. Season was inversely associated with wound infection (p = 0.018) and dehiscence (p = 0.001). Extended LOS was inversely correlated with season (p < 0.001). These relationships held after adjustment. Prolonged LOS (p < 0.001) and any complication (p = 0.108) were more common in the North. CONCLUSIONS: We have demonstrated a graded relationship between seasonality and adverse outcomes following bariatric surgery. The association was strongest for dehiscence and prolonged LOS. These relationships held when using latitude. A prospective study measuring pre-operative 25(OH)D concentration would strengthen the case for causality in adverse surgical outcomes.

Secondary acute myelogenous leukemia with MLL gene rearrangement following radioimmunotherapy (RAIT) for non-Hodgkin's lymphoma.

Targeted therapy with conjugated and unconjugated monoclonal antibodies for non-Hodgkin's lymphoma has revolutionized the approach to this disease. The efficacy and low toxicity of these agents have allowed introduction of this strategy in the early stages of therapy. Longer follow-up is needed before validating the safety of these agents. Since monoclonal antibodies are being given as front-line therapy, it is important to identify all potential adverse events. We report a case of secondary acute myelogenous leukemia (AML) with 11q23 cytogenetic abnormality and mixed lymphoid leukemia (MLL) gene expression in a patient treated with Y90 labeled anti-CD20 antibody (Zevalin). The patient was not exposed to topoisomerase II inhibitors. Our observations suggest a relationship between 11q23 leukemia and radioimmunotherapy (RAIT) and further studies are needed.

Interactions between methylating and pyridyloxobutylating agents in A/J mouse lungs: implications for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis.

The tobacco-specific nitrosamine, 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone, is activated to lung DNA methylating and pyridyloxobutylating intermediates. It is likely that both pathways play a role in lung tumor initiation by this nitrosamine. Previous studies indicated that O(6)-methylguanine (O(6)-mG) persistence is critical for lung tumor formation in A/J mice. The model pyridyloxobutylating agent, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc), enhanced the tumorigenic activity of a model methylating agent, acetoxymethylmethylnitrosamine (AMMN), presumably by increasing O(6)-mG persistence in lung DNA. We have been testing the hypothesis that the pyridyloxobutylation pathway increases the mutagenic activity of the DNA methylation pathway by preventing the repair of O(6)-mG by O(6)-alkylguanine-DNA alkyltransferase (AGT). In this study, we report that NNKOAc depletes AGT in lungs but not livers of A/J mice. The consequences of AGT depletion by NNKOAc were then compared with those observed with a known AGT inhibitor, O(6)-benzylguanine (O(6)-bG). NNKOAc and O(6)-bG had similar effects on the levels of AMMN-derived O(6)-mG at 4 and 96 h postinjection. This increase in O(6)-mG levels correlated to increased lung tumor multiplicity in animals simultaneously treated with AMMN (0.75 or 1 micromol) and NNKOAc or O(6)-bG. Only NNKOAc significantly increased lung tumor multiplicity at doses of 0.25 or 0.5 micromol AMMN. The results from these studies indicate that the pyridyloxobutylating agent, NNKOAc, can influence the tumorigenic activity of methylating agents in two ways. At low AMMN doses, the increase in tumor multiplicity is dominated by the additive tumorigenic properties of AMMN and NNKOAc. At higher AMMN doses, NNKOAc appears to enhance the tumorigenic activity of AMMN through enhanced depletion of the repair protein, AGT, leading to increased O(6)-mG persistence. It is likely that similar interactions are important for the organospecific effects of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone.

Angiogenesis in acute promyelocytic leukemia: induction by vascular endothelial growth factor and inhibition by all-trans retinoic acid.

Recent studies indicate that angiogenesis is important in the pathogenesis of leukemias, apart from its well-established role in solid tumors. In this study, the possible role of angiogenesis in acute promyelocytic leukemia (APL) was explored. Bone marrow trephine biopsies from patients with APL showed significantly increased microvessel density and hot spot density compared with normal control bone marrow biopsies. To identify the mediators of angiogenesis in APL, quantitative and functional assays were performed using the NB4 APL cell line as a model system. Conditioned media (CM) from the NB4 cells strongly stimulated endothelial cell migration. CM from the NB4 cells contained high levels of vascular endothelial growth factor (VEGF) but not basic fibroblast growth factor (bFGF). Most important, the addition of neutralizing VEGF antibodies completely inhibited the ability of NB4 CM to stimulate endothelial cell migration, suggesting that APL angiogenesis is mediated by VEGF. The effect of all-trans retinoic acid (ATRA) on APL angiogenesis was then studied. ATRA therapy resulted in a decrease in bone marrow microvessel density and hot spot density. CM from ATRA-treated APL cells did not stimulate endothelial cell migration. Finally, quantitative assays showed that ATRA treatment resulted in the abrogation of VEGF production by the NB4 cells. These results show that there is increased angiogenesis and VEGF production in APL and that ATRA therapy inhibits VEGF production and suppresses angiogenesis. The addition of specific antiangiogenic agents to differentiation therapy or chemotherapy should be explored. (Blood. 2001;97:3919-3924)

A reactive metabolite of furan, cis-2-butene-1,4-dial, is mutagenic in the Ames assay.

Furan is classified as a nongenotoxic hepatocarcinogen. It is thought to be activated to a toxic metabolite, cis-2-butene-1,4-dial, which is acutely toxic to liver cells. The resulting cytotoxicity is followed by compensatory cell proliferation, increasing the likelihood of tumor production. We examined the genotoxic activity of cis-2-butene-1,4-dial in several strains of Salmonella typhimurium commonly used in the Ames assay. This reactive compound tested positive in TA104, a strain that is sensitive to aldehydes. Mutagenic activity was concentration-dependent (1000 +/- 180 revertants/micromol). Incubation of cis-2-butene-1,4-dial with glutathione prior to addition of bacteria inhibited both the acute toxic and genotoxic activity of this compound. No evidence of mutagenic activity was seen at nontoxic concentrations in TA97, TA98, TA100, and TA102. Our findings are consistent with the hypothesis that cis-2-butene-1,4-dial reacts with DNA to form mutagenic adducts. Our data suggest that cis-2-butene-1,4-dial may be an important genotoxic as well as toxic intermediate in furan-induced tumorigenesis.

Synthesis of DNA oligonucleotides containing site-specifically incorporated O6-[4-oxo-4-(3-pyridyl)butyl]guanine and their reaction with O6-alkylguanine-DNA alkyltransferase.

DNA pyridyloxobutylation occurs during the metabolic activation of the tobacco-specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN). This pathway contributes significantly to the carcinogenic and mutagenic activity of these nitrosamines. In general, the chemical structure of pyridyloxobutyl adducts are not well understood. Recently, an AGT reactive pyridyloxobutyl adduct was identified as O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6-pobG). To better understand the importance of this adduct to the biological activity of pyridyloxobutylating agents, we developed a method for site-specifically incorporating O6-pobG into DNA oligonucleotides. They were synthesized using the phosphoramidite of the precursor 2'-deoxy-O6-{3-[2-(3-pyridyl)-1,3-dithian-2-yl]propyl}guanosine. The dithiane group was oxidatively removed with N-chlorosuccinimide in a final postoligomerization reaction to generate the desired product. Human AGT with a polyhistidine tag was able to repair the O6-pobG-containing DNA oligonucleotide, generating unmodified oligonucleotide. These results are consistent with an alkyl group transfer mechanism for the repair of O6-pobG by AGT.

Metabolism of N-nitrosobenzylmethylamine by human cytochrome P-450 enzymes.

N-Nitrosobenzylmethylamine (NBzMA) is a potent esophageal carcinogen in rodents, and has been found as a dietary contaminant in certain areas of China where esophageal cancer is endemic. To determine which cytochrome P-450 enzymes in humans are primarily responsible for NBzMA metabolism, microsomes from lymphoblastoid cell lines expressing a panel of human cytochrome P-450s (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2D6, CYP2E1, CYP2C9, CYP2C19, and CYP3A4) and a panel of 10 different human liver microsomal preparations were examined for their abilities to metabolize [3H]NBzMA. In addition, the ability of human liver microsomes to form various NBzMA metabolites was correlated with the abilities of these preparations to metabolize coumarin, ethoxyresorufin, chlorzoxazone, 7-ethoxy-4-trifluoromethylcoumarin, S-mephenytoin, and nifedipine. NBzMA metabolites were quantitated by reversed-phase high-performance liquid chromatography (HPLC) coupled with flow-through radioactivity detection. Major metabolites included benzaldehyde, benzyl alcohol, benzoic acid, and several uncharacterized radioactive peaks. Of the representative P-450 activities, only CYP2E1 and CYP2A6 catalyzed substantial metabolism of NBzMA. Compared to CYP2E1, CYP2A6 metabolized NBzMA more readily. NBzMA acted as a potent inhibitor of coumarin 7-hydroxylation in CYP2A6 microsomes. Human liver microsomes metabolized NBzMA readily. NBzMA metabolite formation was most highly correlated with coumarin 7-hydroxylase activity, a marker of CYP2A6 activity. 8-Methoxypsoralen substantially inhibited NBzMA metabolism in human hepatic microsomes. When the effects of the potent isothiocyanates PEITC and PHITC were analyzed on microsomes from cell lines expressing CYP2E1 and CYP2A6, it was found that PEITC inhibited both enzymes, PHITC was the more effective inhibitor of CYP2E1, and PHITC was an ineffective inhibitor of CYP2A6. Collectively, these data indicate that CYP2A6 and, to a lesser degree, CYP2E1 are important P-450 enzymes in the activation of NBzMA in human systems.

An improved method for construction of directionally cloned cDNA libraries from microdissected cells.

Here, we developed an improved method for constructing microdissected cDNA libraries, based on strand-switching properties of reverse transcriptase, followed by PCR amplification with primers to mediate unidirectional insert cloning. Using RNA from microdissected ovarian carcinoma cells, we constructed a cDNA library consisting of 1.3 x 10(6) unidirectional recombinants with an average insert size of 500 bp. Single-pass sequencing of 100 clones with the T7 primer revealed 89 inserts derived from known genes, anonymous expressed sequence tags (ESTs), or novel sequences. Among these clones were known genes and ESTs previously found in cDNA libraries from bulk ovarian tissue RNA, sequences seen for the first time in an ovarian-derived library, and novel sequences not previously seen in any cDNA library. These results demonstrate a methodology for constructing quality cDNA libraries that are cloned in a unidirectional fashion, are complex and diverse, and reflect the tissue of origin.

Evidence for metabolic activation of N'-nitrosonornicotine and N-nitrosobenzylmethylamine by a rat nasal coumarin hydroxylase.

Kinetic parameters were determined for the hydroxylation of N'-nitrosonornicotine (NNN), N-nitrosobenzylmethylamine (NBzMA), coumarin, and ethoxycoumarin catalyzed by rat nasal mucosa microsomes. NNN is a tobacco-specific nitrosamine that, in rats, causes tumors in the nasal cavity and esophagus, whereas NBzMA induces tumors in rat esophagus. Both nitrosamines require alpha-hydroxylation to exert their carcinogenic effects. NNN, NBzMA, coumarin, and ethoxycoumarin were all extensively hydroxylated by rat nasal mucosa microsomes. The KM values for the hydroxylation of each substrate were low, ranging between 2 and 5 microM. 2'- and 5'-Hydroxylation of NNN were catalyzed to a similar extent. NBzMA was metabolized predominantly to benzaldehyde, the product of debenzylation, or methylene hydroxylation. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), NNN, and NBzMA were inhibitors of coumarin and ethoxycoumarin hydroxylation. NNN hydroxylation by nasal mucosa microsomes was inhibited by coumarin, ethoxycoumarin, NNK, and NBzMA but not by N-nitrosodimethylamine. 8-Methoxypsoralen, a potent inhibitor of P450 2A6- and 2a5-dependent coumarin hydroxylation in human and mouse liver microsomes, also significantly inhibited NNN activation. The results of this study suggest that the four substrates examined are hydroxylated by closely related P450 enzymes in rat nasal mucosa and that a coumarin hydroxylase metabolizes both NNN and NBzMA.

Characterization of amino acid and glutathione adducts of cis-2-butene-1,4-dial, a reactive metabolite of furan.

Metabolic activation of the hepatocarcinogen furan yields metabolites that react covalently with proteins. cis-2-Butene-1,4-dial is a microsomal metabolite of furan. This reactive aldehyde is thought to be the toxic metabolite that is responsible for the carcinogenic activity of furan. In order to characterize the chemistry by which this unsaturated dialdehyde could alkylate proteins, the products formed upon reaction of cis-2-butene-1,4-dial with model nucleophiles in pH 7.4 buffer were investigated. N(alpha)-Acetyl-L-lysine (AcLys) reacts with cis-2-butene-1,4-dial to form N-substituted pyrrolin-2-one adducts. N-Acetyl-L-cysteine (AcCys) reacts rapidly with cis-2-butene-1,4-dial to form multiple uncharacterized products. The inclusion of AcLys in this reaction mixture yielded an N-substituted 3-(S-acetylcysteinyl)pyrrole adduct which links the two amino acid residues. Related compounds were isolated when cis-2-butene-1,4-dial and glutathione (GSH) were combined. In this case, cis-2-butene-1,4-dial cross-linked two molecules of GSH resulting in either cyclic or acyclic adducts depending on the relative GSH concentration. Incubation of furan with rat liver microsomes in the presence of [glycine-2-3H]GSH led to the formation of radioactive peaks that coeluted with synthetic standards for the bisgluthathione conjugates. These studies demonstrate that the reactive cis-2-butene-1,4-dial formed during the microsomal oxidation of furan reacts rapidly and completely with amino acid residues to form pyrrole and pyrrolin-2-one derivatives. Therefore, this metabolite is a likely candidate for the activated furan derivative that binds to proteins. The ease with which cis-2-butene-1,4-dial cross-links amino acids suggests that pyrrole-thiol cross-links may be involved in the toxicity observed following furan exposure.

Pyridyloxobutyl adduct O6-[4-oxo-4-(3-pyridyl)butyl]guanine is present in 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone-treated DNA and is a substrate for O6-alkylguanine-DNA alkyltransferase.

The lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is activated to reactive metabolites that methylate or pyridyloxobutylate DNA. Previous studies demonstrated that pyridyloxobutylated DNA interferes with the repair of O6-methylguanine (O6-mG) by O6-alkylguanine-DNA alkyltransferase (AGT). The AGT reactivity of pyridyloxobutylated DNA was attributed to (pyridyloxobutyl)guanine adducts. One potential AGT substrate adduct, 2'-deoxy-O6-[4-oxo-4-(3-pyridyl)butyl]guanosine (O6-pobdG), was prepared. This adduct was stable at pH 7.0 for greater than 13 days and to neutral thermal hydrolysis conditions (pH 7.0, 100 degrees C, 30 min). Under mild acid hydrolysis conditions (0.1 N HCl, 80 degrees C), O6-pobdG was depurinated to yield O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6-pobG). O6-pobdG was hydrolyzed to 4-hydroxy-1-(3-pyridyl)-1-butanone and guanine under strong acid hydrolysis conditions (0.8 N HCl, 80 degrees C). O6-pobG was detected in 0.1 N HCl hydrolysates of DNA alkylated with the model pyridyloxobutylating agent 4-(acetoxymethylnitrosamino)-1-(3-[5-3H]pyridyl)-1-butanone ([5-3H]NNKOAc). When [5-3H]NNKOAc-treated DNA was incubated with either rat liver or recombinant human AGT, O6-pobG was removed, presumably a result of transfer of the pyridyloxobutyl group from the O6-position of guanine to AGT's active site.

Metabolic activation of aromatic amines by human pancreas.

Epidemiologic studies have suggested that aromatic amines (and nitroaromatic hydrocarbons) may be carcinogenic for human pancreas. Pancreatic tissues from 29 organ donors (13 smokers, 16 non-smokers) were examined for their ability to metabolize aromatic amines and other carcinogens. Microsomes showed no activity for cytochrome P450 (P450) 1A2-dependent N-oxidation of 4-aminobiphenyl (ABP) or for the following activities (and associated P450s): aminopyrine N-demethylation and ethylmorphine N-demethylation (P450 3A4); ethoxyresorufin O-deethylation (P450 1A1) and pentoxyresorufin O-dealkylation (P450 2B6); p-nitrophenol hydroxylation and N-nitrosodimethyl-amine N-demethylation (P450 2E1); lauric acid omega-hydroxylation (P450 4A1); and 4-(methylnitrosamino)-1-(3-pyridyl-1-butanol) (NNAL) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) alpha-oxidation (P450 1A2, 2A6, 2D6). Antibodies were used to examine microsomal levels of P450 1A2, 2A6, 2C8/9/18/19, 2E1, 2D6, and 3A3/4/5/7 and epoxide hydrolase. Immunoblots detected only epoxide hydrolase at low levels; P450 levels were <1% of liver. Microsomal benzidine/prostaglandin hydroperoxidation activity was low. In pancreatic cytosols and microsomes, 4-nitrobiphenyl reductase activities were present at levels comparable to human liver. The O-acetyltransferase activity (AcCoA-dependent DNA-binding of [3H]N-hydroxy-ABP) of pancreatic cytosols was high, about twothirds the levels measured in human colon. Cytosols showed high activity for N-acetylation of p-aminobenzoic acid, but not of sulfamethazine, indicating that acetyltransferase-1 (NAT1) is predominantly expressed in this tissue. Cytosolic sulfotransferase was detected at low levels. Using 32P-post-labeling enhanced by butanol extraction, putative arylamine-DNA adducts were detected in most samples. Moreover, in eight of 29 DNA samples, a major adduct was observed that was chromatographically identical to the predominant ABP-DNA adduct, N-(deoxyguanosin-8-yl)-ABP. These results are consistent with a hypothesis that aromatic amines and nitroaromatic hydrocarbons may be involved in the etiology of human pancreatic cancer.

Mechanism of enhancement of esophageal tumorigenesis by 6-phenylhexyl isothiocyanate.

6-Phenylhexyl isothiocyanate (PHITC) enhances esophageal tumorigenesis induced by the carcinogen N-nitrosomethylbenzylamine (NMBA) in rats while its shorter chain analog, phenethyl isothiocyanate (PEITC), inhibits NMBA-induced esophageal tumorigenesis. A significant increase in O6-methylguanine levels in esophageal DNA at 72 h after NMBA administration to rats pretreated with PHITC suggested that PHITC might enhance NMBA metabolic activation or inhibit DNA repair. To test this hypothesis, groups of 20 rats were administered PEITC or PHITC at concentrations of 0, 1.0, or 2.5 mmol/kg in modified AIN-76A diet for 2 weeks. The esophagi were removed from rats, stripped, split, and maintained in HEPES buffered saline (HBS) for assays of NMBA metabolism (n = 5 per group) or were snap frozen for DNA repair assays (n = 15 per group). The principal metabolites of NMBA produced by esophageal explants were: two unidentified peaks, benzyl alcohol (at 4 h only), and benzoic acid. Esophageal explants from PEITC-treated animals showed a significantly decreased ability to metabolize NMBA as expected. PHITC-treated animals showed a slight inhibition in the formation of most NMBA-related metabolites, rather than an overall increase in NMBA activation. This inhibition was less than that observed with PEITC. No inhibitory effects were observed on O6-alkylguanine transferase (AGT) activity in the esophagi of rats treated with 1.0 micromol/g or 2.5 micromol/g PHITC. Thus, effects of PHITC on esophageal metabolism and DNA repair do not account for the enhancement of NMBA tumorigenicity by PHITC.

N-Nitrosobenzylmethylamine is activated to a DNA benzylating agent in rats.

The ability of rat tissues to activate the esophageal carcinogen, N-nitrosobenzylmethylamine (NBzMA), to a DNA benzylating intermediate was investigated. [3-3H]NBzMA was prepared and given to male F344 rats. Tissues were harvested 4 h after treatment, and DNA was isolated. HPLC analysis with radiochemical detection of chemical and enzymatic hydrolysates of DNA from liver and lung revealed the formation of benzyl adducts. Benzyl alcohol, N2-benzylguanine, 3-benzyladenine, N6-benzyladenine, and 7-benzylguanine were the major radioactive components in the hydrolysates. An unknown adduct was also observed. The adduct distribution was similar to that observed in [3-3H]benzylnitrosourea ([3-3H]BzNU)-treated calf thymus DNA. However, enzymatic hydrolysates of [3-3H]BzNU-treated DNA also contained significant levels of O6-benzyl-2'-deoxyguanosine (O6-BzdG). This radioactive adduct disappeared upon incubation of the DNA with a crude preparation of the repair protein, O6-alkylguanine-DNA alkyltransferase isolated from rat liver. These data provide evidence that O6-BzdG is probably rapidly repaired in vivo. No benzylation of esophageal mucosal DNA was detected. The level of DNA benzylation observed in tissues from [3-3H]NBzMA-treated rats was several orders of magnitude lower than the level of DNA methylation in these same tissues. Therefore, these data indicate that DNA benzylation plays a minor role, if any, in the carcinogenic activity of NBzMA.

What proportion of primary psychiatric interventions are based on evidence from randomised controlled trials?

OBJECTIVES: To estimate the proportion of psychiatric inpatients receiving primary interventions based on randomised controlled trials or systematic reviews of randomised controlled trials. DESIGN: Retrospective survey. SETTING: Acute adult general psychiatric ward. SUBJECTS: All patients admitted to the ward during a 28 day period. MAIN OUTCOME MEASURES: Primary interventions were classified according to whether or not they were supported by evidence from randomised controlled trials or systematic reviews. RESULTS: The primary interventions received by 26/40 (65%; 95% confidence interval (95% CI) 51% to 79%) of patients admitted during the period were based on randomised trials or systematic reviews. CONCLUSIONS: When patients were used as the denominator, most primary interventions given in acute general psychiatry were based on experimental evidence. The evidence was difficult to locate; there is an urgent need for systematic reviews of randomised controlled trials in this area.

Diethylnitrosamine exposure-responses for DNA damage, centrilobular cytotoxicity, cell proliferation and carcinogenesis in rat liver exhibit some non-linearities.

The exposure-responses for several effects of limited exposures to diethylnitrosamine (DEN) in the livers of male Fischer 344 rats were measured and phenobarbital promotion was used to enhance expression of initiation of carcinogenesis. Five doses ranging from a cumulative total of 0.5 to 4 mmol DEN per kg body weight were given as weekly i.p. injections for 10 weeks. This was followed by 4 weeks recovery, after which the groups were maintained on either a basal diet or 0.05% phenobarbital (PB) to promote liver tumor development. All doses of DEN produced ethylation in liver DNA, which increased with dose. Indicative of toxicity, the centrilobular zone of glutamine synthetase-positive hepatocytes was reduced in relationship to exposure up to a cumulative exposure of 3 mmol/kg. The two lower exposures to DEN produced no increase in cell proliferation, whereas higher exposures resulted in marked increases, approximately 4-fold between 1.0 and 2.0 mmol/kg cumulative. At the end of the recovery period (14 weeks), hepatocellular altered foci (HAF), which expressed the placental form of glutathione S-transferase, were induced by all exposures, with an increase of approximately 4-fold between the exposures of 1.0 and 2.0 mmol/kg being the greatest. In rats maintained on basal diet or PB for 24 weeks after exposure, HAF increased further and with exposures of 2.0 mmol/kg and above, all rats developed hepatocellular carcinomas. With 1.0 mmol/kg, no liver tumor occurred in 12 rats without promotion, whereas in those given PB, two adenomas and two carcinomas were present in 12 rats. At the lowest exposure of 0.5 mmol/ kg, no tumor occurred in rats on basal diet, although HAF increased approximately 7-fold. With PB promotion, only one adenoma developed in 12 rats and HAF increased another 2-fold. Thus, the findings document non-linearity for some of the effects of DEN and a near no-effect level for initiation of promotable liver neoplasms at the lowest exposure in spite of a substantial induction of HAF.

Pyridyloxobutylation of guanine residues by 4-[(acetoxymethyl)nitrosamino]-1-(3-pyridyl)-1-butanone generates substrates of O6-alkylguanine-DNA alkyltransferase.

Pyridyloxobutylation of DNA yields adducts that react with O6-alkylguanine-DNA alkyl-transferase (AGT) to prevent the repair of O6-methylguanine (O6-mG). The chemical characterization of pyridyloxobutyl adducts has been confounded by their instability under DNA hydrolysis conditions. They decompose to 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) during the chemical or enzymatic hydrolysis of DNA. The goal of these studies was to determine which bases are pyridyloxobutylated to form AGT-reactive adducts. The model pyridyloxobutylating agent, 4-[(acetoxymethyl)nitrosamino]-1-(3-pyridyl)-1-butanone (NNKOAc), was reacted with either poly(dAdT) or poly(dGdC) to generate DNA substrates for reaction with AGT. Only the pyridyloxobutylated poly(dGdC) was able to prevent the ability of partially purified rat liver AGT to repair O6-mG. These results paralleled those obtained for the corresponding methylated substrates. These studies are consistent with the pyridyloxobutylation of GC base pairs and not AT base pairs in the DNA to generate a substrate for AGT. In order to distinguish between the formation of reactive adducts at C residues versus G residues, two oligomers were designed that were complementary to one another. One oligomer contained A, T, and G residues, whereas its complement contained T, A, and C residues. Only the dG-containing oligomer reacted with NNKOAc to generate an AGT-reactive adduct, again paralleling the results obtained for a methylating agent. These results demonstrate that pyridyloxobutylation of only guanine residues produces adducts that react with AGT. These AGT-reactive guanine adducts are relatively stable within DNA, with a half-life of 1-2 weeks at 37 degrees C. They represent up to 70% of the total HPB-releasing adducts in the NNKOAc-treated DNA. We postulate that a potential AGT-reactive adduct is an O6-(pyridyloxobutyl)guanine adduct.

Effects of dietary fat content on the metabolism of NNK and on DNA methylation induced by NNK.

The available data support the concept that high-fat diets increase cytochrome P-450 activities in the liver, leading to increased rates of carcinogen metabolism and, in some instances, DNA adduct formation. Therefore we investigated whether a high-fat diet can also influence DNA methylation by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the lungs of rats. Male F344 rats were fed a regular AIN-76A low-fat (5% corn oil) or AIN-76A high-fat (23.5% corn oil) diet. After three weeks on this dietary regimen, the animals were injected subcutaneously once daily for four days with NNK at 0.39 mmol/kg body wt. Groups of rats were sacrificed 4 and 24 hours after the last NNK administration; livers and lungs were excised for DNA isolation. We found that the high-fat diet significantly enhanced the formation of O6-methylguanine (O6-mGua) in the rat lung four hours (p < 0.01) after the last carcinogen administration. This may, in part, account for our previous finding in regard to the enhancing effect of the high-fat diet on NNK-induced lung carcinogenesis. There was no effect on O6-mGua or 7-mGua in the rat liver at either time point. To further elucidate the enhancing effect of the high-fat diet on DNA methylation by NNK in the lung, we determined its effect on the in vitro and in vivo metabolism of NNK. The in vitro data indicated that dietary fat has no measurable effect on liver and lung microsomal mixed-function oxidase in catalyzing the metabolic activation of NNK. The results of the metabolism study of NNK in vivo appear to be consistent with the in vitro finding, in that fat had no effect on the excretion pattern of NNK or on the distribution pattern of its urinary metabolites. It is apparent that the enhancing effect of the high-fat diet on O6-mGua in the lung of rats that was measured four hours after NNK injection requires future investigations.

Identification of cis-2-butene-1,4-dial as a microsomal metabolite of furan.

The hepatocarcinogen furan is believed to be activated to the reactive aldehyde, cis-2-butene-1,4-dial, by microsomal enzymes. The rat liver microsomal metabolism of furan was examined in the presence of NADPH and semicarbazide. HPLC analysis of incubation mixtures revealed the formation of a metabolite that coeluted with standards for the bis-semicarbazone adduct of cis-2-butene-1,4-dial. The formation of this compound required the presence of NADPH, semicarbazide, and microsomes. Preparative isolation and chemical characterization of this metabolite confirmed the structural assignment. These data provide evidence that the reactive aldehyde, cis-2-butene-1,4-dial, is a major metabolic product of furan.