Organ specificity in the microsomal activation and toxicity of N-nitrosomethylbenzylamine in various species

The microsomal metabolism of the rat esophageal carcinogen N-nitrosomethylbenzylamine (NMBZA) at the methylene carbon atom to yield benzaldehyde was studied in various organs of a number of species to determine the role of metabolic activation in the carcinogenicity or toxicity of the nitrosamine. In the Sprague-Dawley rat, NMBZA was metabolized by microsomes from liver, lung, and esophageal mucosa. In the F344 rat and rabbit, metabolic activity was present in both liver and esophageal mucosa, the only tissues studied in these species. In contrast, in the Syrian hamster and BALB/cByJ mouse, NMBZA debenzylation was undetectable in the esophagus but occurred at relatively high rates in liver, lung, and kidney. The forestomach mucosa exhibited undetectable levels of activity in the Sprague-Dawley rat and BALB/cByJ mouse, although in the hamster, it was present at a very low level. Administration of a dose of NMBZA acutely toxic to the rat (18 mg/kg i.p.) resulted in significant cellular damage only to the rat esophageal mucosa, no other tissues examined in the rat, hamster, or mouse being affected. These observations, together with the available data on carcinogenicity of the nitrosamine in the rat and rabbit, suggest that in the esophagus, at least, metabolic activation of NMBZA is necessary to elicit its toxic and/or carcinogenic effect. However, NMBZA is also metabolized at a high rate in the liver of all species but is not toxic or carcinogenic in this tissue, suggesting that other factors besides metabolic activation must be involved in the resistance of hepatocytes to the effects of the nitrosamine. Microsomes prepared from human esophageal mucosa from six patients metabolized NMBZA at rates that were either undetectable or approximately 70 times lower than in the Sprague-Dawley rat.     

Substrate concentration dependency of N-nitrosodimethylamine and N-nitrosomethylbenzylamine metabolism in rat liver

Metabolism of N-nitrosodimethylamine (NDMA), a hepatocarcinogen,and N-nitrosomethylbenzylamine (NMBeA), an esophageal carcinogen,was comparatively investigated in rat liver. When these nitrosamines(25 µmole/kg) were administered orally to rats, the clearanceof NDMA from the serum and liver was faster than that of NMBeA.The metabolic decomposition of NDMA by rat isolated hepatocyteswas slower than that of NMBeA at high concentration (0.5 mM).However, at low concentration (6.7 µM) the metabolic decompositionof NDMA was faster than that of NMBeA. The ratio of NDMA demethylationto NMBeA demethylation and debenzylation by hepatic microsomesalso changed depending on nitrosamine concentrations (1 µMto 1 mM), and low concentration of NDMA was demethylated rapidly.These results suggest that NDMA is metabolized to a methylatingagent more effectively than NMBeA in liver, when carcinogenicdoses of nitrosamines are administered.     

Alpha-glucuronides of N-nitrosomethylbenzylamine

After administration of N-nitroso[14C-methyl]benzylamine to rats, a glucuronide was demonstrated in the urine, the structure of which could be assigned on the basis of synthetic marker compounds. These findings offer an explanation for the failure to detect benzylation of DNA after administration of N-nitrosomethylbenzylamine (NMBzA). The glucuronides were synthesized by a combination of enzymatic and chemical reactions.     

Extrahepatic microsomal metabolism of N-nitrosodi-n-butylamine in rats

The omega- and omega-1-hydroxylation of N-nitrosodi-n-butylamine (NDBA) has been studied in microsomes from rat liver, lung, intestine and kidney. Both reactions followed at least two enzyme kinetics with low (2-10 microM) and high (1 mM) Km values. Whereas omega-1-hydroxylation was the predominant pathway in liver, omega-hydroxylation was more important in extrahepatic tissues. First-pass metabolism of NDBA in lungs and intestinal mucosa may be of importance in the development of urinary bladder tumours in rats.     

Dietary ellagic acid reduces the esophageal microsomal metabolism of methylbenzylnitrosamine

Dietary ellagic acid has been shown to reduce the incidence of methylbenzylnitrosamine-induced esophageal carcinoma in the rat. Methylbenzylnitrosamine (MBN) is a naturally occurring carcinogen which requires cytochrome P-450 dependent activation to be mutagenic. We examined whether the reduction in tumor incidence observed with dietary ellagic acid was associated with alterations in the cytochrome P-450 dependent microsomal metabolism of MBN. Dietary ellagic acid was shown to significantly reduce total esophageal and hepatic microsomal cytochrome P-450 (P less than 0.05) and significantly reduce the esophageal microsomal metabolism of MBN (P less than 0.05). The addition of ellagic acid in vitro also resulted in a significant inhibition (P less than 0.05) of the esophageal microsomal metabolism of MBN. In contrast, dietary ellagic acid and the addition of ellagic acid in vitro did not alter the hepatic microsomal metabolism of MBN. The reduced rate of MBN metabolism by the esophageal microsomes from the ellagic acid fed rats may contribute to the decreased incidence of esophageal carcinoma observed in these animals.     

Tissue, sex, and animal species specificity of aflatoxin B1 inhibition of nuclear RNA polymerase II activity

Recent studies from this laboratory have shown that severalchemical carcinogens, i.e., aflatoxin B₁, N-OH-2-acetylaminofluorene,actinomycin D, and methylazoxymethanol acetate, when administeredin vivo, have all produced a selective and dramatic inhibitionof rat liver nuclear RNA polymerase II activity. To determinewhether this inhibition is related to carcinogenesis, aflatoxinB₁ is used as a model system to test tissue, sex, and animalspecies specificity that is known to be characteristic of carcinogenesis.The results show that aflatoxin B₁ (3 mg/kg body weight, i.p.,2 h) inhibits RNA polymerase II activity only in the targettissue, liver, and not in the non-target tissues, e.g., lungand brain. It inhibits liver RNA polymerase II activity preferentiallyin male over female rats, and has no effect on mouse liver RNApolymerase II activity. These results are in good agreementwith the specificities of aflatoxin B₁ carcinogenesis in thewhole animal systems. Furthermore, with the four principal aflatoxinstested, the order of inhibitory effect on RNA polymerase IIis: B₁ > G₁ > B₂, G₂. It is concluded, therefore, thatthe inhibition of RNA polymerase II activity and carcinogenesisare likely to be related and that it is theoretically soundto use this inhibition as a diagnostic tool to screen potentialcarcinogens.     

Tissue specificity of the action of bleomycin. Autoradiographic studies

The proliferative activity of different types of murine tissue up to 72 h after a single dose of bleomycin was studied via histoautoradiography to determine whether bleomycin has a tissue-specific effect. Bleomycin apparently not only has a general cytotoxic and/or cytostatic action, the strongest being on tissue with a high proliferation rate, but also, as a comparison of the effect of bleomycin on the proliferative activity of squamous epithelium (plantar region, tail, skin, esophagus, tongue) and of mucosal epithelium (stomach, ileum, colon) has indicated, the proliferative activity of squamous epithelium is impaired to a greater extent. This, therefore, distinguishes bleomycin from many other substances used in tumor therapy.     

Bioactivation of N-nitrosomethylbenzylamine and N-nitrosomethyl-amylamine in oesophageal papillomas

Oesophageal papillomas were induced in male F344 rats by continuous exposure to N-nitrosomethylbenzylamine (NMBzA) and N-nitrosomethyl(2-methylbutyl)amine in the drinking water at concentrations of 10 and 19.5 p.p.m. respectively. After 81-141 days animals received a single i.p. chasing dose of NMBzA (0.1 mmol/kg), [14C-methyl]NMBzA or N-nitroso[14C-methyl]amylamine and were killed 6 h later. Induced papillomas (3-9 per animal) were analysed by autoradiography and by immunohistochemistry using a polyclonal antibody to O6-methyldeoxyguanosine. Both techniques revealed the presence of high levels of alkylation products in all papillomas investigated. Immunohistochemical staining of O6-methyldeoxyguanosine was largely restricted to nuclei of the basal layer and of epithelial cells with incipient keratinization. These findings demonstrate that NMBzA and N-nitrosomethylamylamine and probably related methylalkylnitrosamines are effectively bioactivated in premalignant lesions, indicating that during chronic exposure papillomas can acquire additional mutations that are likely to play a major role in tumour progression.     

Effects of dietary N-(4-hydroxyphenyl)retinamide on N-nitrosomethylbenzylamine metabolism and esophageal tumorigenesis in the Fischer 344 rat.

BACKGROUND: 9-cis-Retinoic acid (9-cis-RA) and N-(4-hydroxyphenyl)retinamide (4-HPR) are effective chemopreventive agents against epithelial tumors in the oral cavity, breast, and prostate. We tested the inhibitory activity of these retinoids against N-nitrosomethylbenzylamine (NMBA)-induced tumorigenesis in the rat esophagus. METHODS: Male Fischer 344 rats were randomly assigned to receive diets either lacking or containing 9-cis-RA or 4-HPR for 1 week before tumor initiation with NMBA and then for the duration of the study. NMBA metabolism, O(6)-methylguanine adduct formation, and cytochrome P450 messenger RNA (mRNA) expression in the esophagi of the rats were studied to investigate the mechanisms by which dietary 4-HPR affects tumorigenesis. All statistical tests were two-sided. RESULTS: Dietary 4-HPR resulted in a dose-dependent and statistically significant enhancement (P<.05) of tumorigenesis in response to NMBA. In two different tumor bioassays, the mean tumor multiplicity for rats fed the highest concentration of dietary 4-HPR (0.8 g/kg diet) was increased by 5.9 tumors (95% confidence interval [CI] = 1.7 to 10.1 tumors) and 6.7 tumors (95% CI = 5.6 to 7.8 tumors) compared with the mean tumor multiplicity for rats that received the control diet lacking 4-HPR. Animals fed diets containing 9-cis-RA displayed no statistically significant increase in tumorigenesis. Compared with animals fed a diet lacking 4-HPR, animals fed 4-HPR had increased NMBA metabolism in esophageal explant cultures and had higher levels of O(6)-methylguanine DNA adducts and CYP2A3 mRNA in their esophagi. CONCLUSIONS: Dietary 4-HPR enhances tumorigenesis in response to NMBA in the rat esophagus by increasing tumor initiation events. Dietary 4-HPR may exert paradoxical effects at some sites, such as the aerodigestive tract, by modulating the bioactivation of carcinogens in target tissues.     

Substrate specificity and alkyl group selectivity in the metabolism of N-nitrosodialkylamines

Metabolic activation may be a key step in determining the tissue specificity of carcinogenic nitrosamines. In previous work, we characterized P450IIE1 (an acetone/ethanol-inducible form of cytochrome P-450) as the major enzyme for the metabolic activation of N-nitrosodimethylamine. In this work, we investigated the metabolism of other N-nitrosodialkylamines in rat liver microsomes and in reconstituted monooxygenase systems containing purified cytochrome P-450 isozymes. The enzyme specificities in the metabolism of N-nitrosoethylmethylamine and N-nitrosodiethylamine were similar to those of N-nitrosodimethylamine; i.e., these substrates were more efficiently metabolized by acetone- or ethanol-induced microsomes than by other types of microsomes. However, substituting one methyl group with a benzyl or butyl group, as in N-nitrosobenzylmethylamine or N-nitrosobutylmethylamine (NBMA), substantially changed the enzyme specificity. P450IIE1 efficiently catalyzed the demethylation but not the debutylation of NBMA, whereas P450IIB1 (a phenobarbital-inducible form) efficiently catalyzed both the debutylation and demethylation reactions. In the demethylation of NBMA by P450IIE1, the addition of cytochrome b5 markedly increased the activity at low but not at high substrate concentrations, suggesting a decrease in Km value. This effect, however, was not observed in the debutylation of NBMA by P450IIE1 or P450IIB1, and in the demethylation of NBMA by P450IIB1. These studies demonstrate the substrate specificity and alkyl group selectivity in the metabolism of nitrosamines by cytochrome P-450 isozymes.     

Effects of the induction of hepatic microsomal metabolism on the toxicity of cyclophosphamide

Cyclophosphamide (CP) administration to rats in a single i.p. dose (200 mg kg-1), while producing urinary bladder toxicity and 30-40% depression of the hepatic microsomal mixed function oxidase (MFO), failed to produce any depression of MFO activities in extrahepatic tissues such as lung, kidney and intestine. Phenobarbital pretreatment of the rats, which is known to enhance hepatic microsomal activation of CP, protected against CP-induced urinary bladder toxicity and the depression of hepatic MFO activities. This protection appears to be, at least in part, related to phenobarbital induction of hepatic cytochrome P-450 isozyme(s) that metabolizes CP to a new metabolite tentatively identified as didechlorodihydroxycyclophosphamide.     

Potent inhibition of oesophageal metabolism of N-nitrosomethylbenzylamine, an oesophageal carcinogen, by higher alcohols present in alcoholic beverages.

The main cause of oesophageal cancer in western countries is consumption of alcoholic beverages, the degree of risk being much greater for certain spirits than for wine or beer. Risk shows a striking correlation with the content of higher alcohols in the drinks, although the alcohols per se have not been shown to be carcinogenic in experimental animals. To test the concept that higher alcohols modulate the oesophageal carcinogenicity of nitrosamines by altering their metabolism, we studied the effect of certain higher alcohols on the metabolism of N-nitrosomethylbenzylamine by rat oesophageal mucosal and liver microsomes. In oesophagus, the alcohols were 1000 times more inhibitory than ethanol, and in liver 100 times. This suggests that enhancement of carcinogenesis may not result from an effect on nitrosamine metabolism. Higher alcohols could act by increasing the rate of replication of cells already initiated for malignancy by previous exposure to nitrosamines. Intubation of 2-methylbutanol produces a very much greater increase in oesophageal basal-cell proliferation than does ethanol.     

The metabolism of 9,10-dimethylanthracene by rat liver microsomal preparations

The metabolism of the weakly-carcinogenic hydrocarbon, 9,l0-dimethylanthracene(DMA) by rat-liver microsomal preparations has been examined.9-Hydroxymethyl-10-methylanthracene (9-OHMeMA) and 9,10-dihydroxymethyl-anthracene(9,l0-DIOHMeA) were identified as metabolites .by comparingtheir chromatographic and spectral properties with those ofthe authentic compounds. The trans-1,2-dihydro-1,2-dihydroxyderivative of DMA (DMA 1,2-diol) was the major metabolite formedwhich was identified by its chromatographic, u.v., n.m.r. andmass spectral properties. The dihydrodiol was also formed inthe oxidation of DMA in an ascorbic acid-ferrous sulphate-EDTAsystem. Two other dihydrodiol that were formed from DMA by metabolismappeared to be the trans-1,2-and 3,4-dihydrodiols of 9-OHMeMA(9-OHMeMA 1,2-diol and 9-OHMeMA 3,4-diol) and the further metabolismof DMA 1,2-diol yielded both of these dihydrodiols. When 9-OHMeMAwas further metabolized, two main metabolites were formed; onewas identified as 9,10-DiOHMeA and the other appeared to be9-OHMeMA 3,4-diol. No metabolites were detected when 9,10-DiOHMeAwas incubated with rat-liver microsomal fractions.     

The influence of various enzyme inducers on the microsomal metabolism of 7,8-benzoflavone

Comparative in vitro metabolic experiments with 7,8-benzoflavoneusing microsomes prepared from the livers of control Sprague-Dawleyrats or from rats of the same strain treated with phenobarbitone,3-methylcholanthrene, or Aroclor 1254 (known inducers of hepaticcytochrome P450 mixed function oxidases) disclosed variableamounts of the three metabolites previously reported, namelythe 5,6-epoxide, 9,0-dihydro-9, 10-diol, and the 5 (or 6)-phenol.Microsomes from phenobarbitone-treated rats gave in additiona small amount of the 5,6-dihydro-5,6-diol, identified by massspectrometry and u.v. spectroscopy. The apparent discrepancybetween our previous results (1) and those of Nesnow and Bergman(2) is therefore partly resolved, although the absence of the9,10-diol from their products remains unexplained, but may haveresulted from their use of rats of a different strain.     

Effects of dietary fat on hepatic microsomal metabolism of 1,2-dimethylhydrazine

The present study was designed to examine the effects of different high fat diets on the liver microsomal metabolism of aminopyrine (AMP) and 1,2-dimethylhydrazine (DMH). Male Sprague-Dawley rats were fed either a low fat (5% corn oil) or high fat (20%) diets containing either corn oil, menhaden oil or beef tallow for a period of up to 9 months. Liver microsomes were assayed for N-demethylase activity for both AMP and DMH substrates at 2 weeks, 1, 6 and 9 months of diet only, and also after 1, 2, 5 and 10 DMH treatments (20 mg/kg body weight). The menhaden oil-fed group had consistently higher AMP demethylase activity, which increased up to 6 months and then declined. Beef tallow-fed rats had the highest DMH demethylase activity following DMH, but this decreased by 10 treatments. These data indicate that type and amount of dietary fat affects microsomal metabolism of carcinogens, which may enhance tumor initiation.     

Oxidative microsomal metabolism of 1-nitropyrene and DNA-binding of oxidized metabolites following nitroreduction

1-Nitropyrene is an environmental mutagen and carcinogen whichundergoes both oxidative and reductive metabolism. We have previouslyshown that nitroreduction to N-hydroxy-1-aminopyrene leads tothe formation of arylamine-DNA adducts. In the present study,we have investigated the oxidative metabolism of 1-nitropyreneand the subsequent binding of ring-oxidized metabolites to DNA.In vitro incubations were conducted using hepatic microsomesfrom uninduced rats or from rats pretreated with phenobar-bital,Aroclor 1254, 3-methykholanthrene, or 3-methyl-cholanthreneand trans-stilbene oxide. H.p.l.c. analysis of the incubationmixtures indicated the presence of the previously reported metabolites,1-aminopyrene, 3-, 6-, and 8-hydroxy-1-nitropyrene, and 1-nitropyrenetrans-4, 5-dihydrodiol. In addition, 1-nitropyrene 4, 5-oxide,1-nitropyrene 9, 10-oxide, 1-nitropyrene ira/is-9, 10-dihydrodioland 1-pyrenol were identified. The formation of both K-regiondihydrodiols could be increased by transstHbene oxide inductionof microsomal epoxide hydrase. Formation of the K-region epoxideswas greatest using phenobarbital- and Aroclor-induced microsomesand increased with increasing oxygen tension, while 1-pyrenolformation was highest in 3-methylcholanthrene-induced microsomalincubations and was not affected by the oxygen tension. Whencalf thymus DNA was added to the microsomal incubations, similarlevels of DNA-binding occurred in incubations conducted underoxygen, air, argon or anaerobic conditions. H.p.l.c. analysisof the enzymatically hydrolyzed DNA indicated the presence ofmultiple DNA adducts with the major product coeluting with AKdeoxy-guanosin-8-yl)-l-aminopyrene.The K-region oxides bound directly to DNA to give adducts similarto the minor products detected in the microsomal incubations.Incubation of the K-region oxides with the nitroreductase, xanthineoxidase, increased the DNA-binding and resulted in an additionalad-duct which coeluted with AKdeoxyguanosin-8-yl)-l-amino pyrene.3-Hydroxy-l-nitropyrene bound extensively to DNA upon nitroreductionby rat liver cytosol or xanthine oxidase, while 6- and 8-hydroxy-l-nitropyrenebound only slightly. None of these oxidized metabolites wasactivated to DNA-binding species by cytosolic nitroreductionfollowed by AcCoA-dependent acetylation. The fact that oxidizedmetabolitesof 1-nitropyrene are reduced to DNA-binding derivatives moreeasily than 1-nitropyrene itself may be important in vivo where1-nitropyrene has been shown to be readily oxidized.     

中国人尿路斑块蛋白Ib组织特异性及其启动子的克隆与鉴定

目的:研究中国人尿路斑块蛋白Ib的组织特异性并克隆与鉴定其启动子。方法:RT-PCR检测32例膀胱癌、16例正常膀胱粘膜、15例正常小肠粘膜、16例正常食道粘膜、19例正常肾实质、20例正常肝实质、8例正常皮肤及8例正常心肌标本中尿路斑块蛋白Ib的表达。以人正常膀胱粘膜为材料提取基因组DNA,克隆尿路斑块蛋白Ib启动子片段,并凝胶电泳和测序鉴定。结果:所有正常膀胱粘膜标本、16例分化Ⅰ级膀胱癌中有15例(93.8%)、9例分化Ⅱ级膀胱癌中有7例(77.8%)、7例分化Ⅲ级膀胱癌中有4例(57.1%)检测到尿路斑块蛋白Ib表达,而正常食道粘膜、小肠粘膜、肾实质、肝实质、皮肤、心肌标本均未检测到尿路斑块蛋白Ib表达。中国人尿路斑块蛋白Ib234bp的启动子被克隆并完成鉴定。结论:中国人尿路斑块蛋白Ib具有高度尿路移行上皮特异性,加上其启动子短小,尿路斑块蛋白Ib启动子非常适合膀胱癌的靶向基因治疗研究。我们成功克隆出中国人尿路斑块蛋白Ib启动子片段,为后续研究奠定了基础。     

Preferential methylation of target organ DNA by the oesophageal carcinogen N-nitrosomethylbenzylamine

Male Wistar rats received a single i.v. injection of the oesophageal carcinogen N-nitroso[methyl-14C]-methylbenzylnitrosamine (2.5 mg/kg body weight). Rapid distribution of the carcinogen occurred, with highest initial concentrations in liver and kidney. Within 10 min after the injection, 14C-labelled metabolites accounted for 50% of the total radioactivity present in the oesophagus, for approximately 25% in liver and forestomach, and for less than 20% in all other organs investigated. Decay of the carcinogen in rat serum followed first-order kinetics with a half-life of 35 min. Of the total radioactivity administered, 49% was exhaled as 14CO2 within 10 h and an additional 5-10% was excreted via urine and faeces. Four hours after a single i.v. injection of N-nitroso-[methyl-14C]benzylnitrosamine methylation of purine bases in DNA was most extensive in the oesophagus, followed by liver, lung and forestomach DNA. In the remaining tissues, DNA methylation was either considerably less (kidney, glandular stomach, spleen) or not at all detectable (ileum, colon, brain). At this time the concentration of the promutagenic base O6-methylguanine in oesophageal DNA was six times higher than in lung and nine times higher than in hepatic DNA. These data suggest that in the rat the selective induction of oesophageal tumours by N-nitrosomethylbenzylamine and related asymmetrical nitrosamines is mediated by a preferential bioactivation of the carcinogen in the target organ.     

Alterations of microsomal monooxygenase system and carcinogen metabolism by streptozotocin-induced diabetes in rats

The effects of diabetes on the liver microsomai monooxy-genaseenzymes and carcinogen metabolism have been studied in rats.Treatment with streptozotocin causes a marked enhancement inmicrosomai N-nitrosodimethylamine (NDMA) demethylase activity.The enhancement is due mainly to the induction of a high affinityNDMA demethylase (K_m, {small tilde}0.05 mM) which is accompaniedby the induction of a protein species with mol. wt. of 50 000.The treatment also induces aniline hydroxylase whose activityis in parallel with NDMA demethylase. Streptozotocin-induceddiabetes also increases the metabolism of N-nitrosomethyl-ethylaminebut not that of N-nitrosomethylaniline or N-nitrosomethylbenzylamine.On the other hand, diabetes decreases the metabolism of benzo[a]pyrene,benzphetamine, and ethylmorphine. The results suggest that diabetescauses an alteration of the composition of cytochrome P-450isozymes; the forms efficient in metabolizing NDMA are increasedwhile certain other forms of cytochrome P-450 are decreased.