Effects of ethanol treatment on DNA damage induced in Escherichia coli K-12 in various organs of mice by N-nitro-sonornicotine, 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone and N-nitrosopyrrolidine

DNA damage induced by tobacco-related nitrosamines was quantitativelydetermined in animal-mediated DNA-repair assays with Escherichiacoli K-12 strains. Intraperitoneal administration of N-nitrosonornicotine(NNN), 4-(methyl-nitrosamino)–1-(3-pyridyl)-l-butanone(NNK) and N-nitro-sopyrrolidine (NPYR) caused dose-dependentgenotoxic effects in indicator bacteria recovered from variousorgans of nitrosamine-pretreated mice. Oral administration ofethanol (6.3 g/kg body wt) 1 h prior to administration of NNN,NNK or NPYR resulted in a substantial reduction of genotoxicitythat was more pronounced in the liver as compared to lungs,spleen, kidneys and blood. However, when the same ethanol dosewas given 26 h before NNN or NPYR, an increase of DNA damagewas found, that was, in most cases, higher in the kidneys thanin the liver. Significant enhancement of genotoxic activitywas also measured in lungs and spleen, whereas only a marginalincrease was detectable in the blood. Repeated administrationof smaller ethanol doses (2.0 g/kg body wt at 12 h intervals)for 4 days caused a comparable increase. Similar enhancementof genotoxicity was also measured when acetone (3.5 g/kg) wasgiven orally 15 h before the nitrosamine administration. Thestimulating effect of ethanol was dose dependent and was absentwhen the alcohol was administered 60 h prior to the nitrosamine.Neither ethanol nor acetone had an effect on the genotoxicityof NNK under identical experimental conditions. The same E.coliK–12 strains were used to test NNN, NNK and NPYR in invitro assays. The ranking order of activation capacity was liverS–9 > kidney S–9 > lung S-9 for all threenitrosamines. Blood S-9 did not markedly activate the nitrosamines.S–9 mixtures prepared from mice that had been treatedwith ethanol (6.3 g/ kg body wt) for 26 h before death activatedNNN and NPYR more efficiently than those S-9s from untreatedanimals. The increase of genotoxic activity was more pronouncedwith S-9 from kidneys and lungs than from liver. No differencewas seen with S–9 from ethanol-treated and untreated micewith NNK.     

Anti Genotoxic Effect of Mosinone-A on 7, 12-Dimethyl Benz[a] Anthracene Induced Genotoxicity in Male Golden Syrian Hamsters

The present study was aimed to evaluate the antigenotoxic effect of Mosinone-A on 7,12-dimethylbenz[a]anthracene induced genotoxicity. The frequency of micronucleated polychromatic erythrocytes [MnPCEs], chromosomal aberrations [CA], DNA damage (comet assay) as cytogenetic markers and the status of lipid peroxidation byproducts, antioxidants and phase II detoxification agents were used as biochemical markers to assess the antigenotoxic effect of Mosinone-A on DMBA induced genotoxicity. A single intraperitoneal injection of DMBA (30 mg/kg b.wt) to golden Syrian hamsters, resulted in marked elevation in the frequency of MnPCEs, aberrations in the chromosomal structure were found in bone marrow and DNA damage (comet assay) was found in blood cells and altered level of lipid peroxidation, antioxidants, and phase II detoxification agents. Oral pretreatment of Mosinone-A (2 mg/kg b.wt) for 5 days to DMBA treated animals significantly reduced the frequency of MnPCEs, chromosomal abnormalities such as chromosomal break, gap, minute, fragment, DNA damage and reversed the status of biochemical variables. Our results thus demonstrated the antigenotoxic effect of Mosinone-A on DMBA induced genotoxicity in male golden Syrian hamsters.     

Occurrence of volatile nitrosamines in food samples collected in three high-risk areas for nasopharyngeal carcinoma

Fifty-four samples of foods consumed frequently in Tunisia, southern China and Greenland, all high-risk areas for nasopharyngeal carcinoma (NPC), were analysed for the presence of volatile nitrosamines by gas chromatography (GC) combined with a thermal energy analyser (TEA). Relatively high levels of N-nitrosodimethylamine (NDMA), N-nitrosopiperidine (NPIP) and N-nitrosopyrrolidine (NPYR) were found in Tunisian stewing base (touklia) and dried mutton preserved in olive oil (qaddid). In one Chinese salted and dried fish sample, a high level of NDMA (133 micrograms/kg) was detected, but for the 14 others the levels ranged from undetectable to 14 micrograms/kg, with a mean of 3 micrograms/kg. Similarly high levels of NDMA, were found in Eskimo dried, unsalted fish samples. NDMA, NPIP and NPYR at various levels were present in Chinese vegetables fermented in brine. The possible role of nitrosamines in the etiology of NPC is discussed.     

Volatile nitrosamine levels in common foods from Tunisia, south China and Greenland, high-risk areas for nasopharyngeal carcinoma (NPC)

On the basis of anthropological pilot studies on diet in Tunisia, south China and Greenland, food items consumed frequently by these populations at high risk for NPC were analysed for volatile nitrosamines using gas chromatography combined with a thermal energy analyzer. Relatively high levels of N-nitrosodimethylamine (NDMA), N-nitrosopyrrolidine (NPYR) and N-nitrosopiperidine (NPIP) were detected in Tunisian stewing base ("Touklia") and dried mutton preserved in olive oil ("Qaddid"). NDMA was also detected at levels ranging from trace amounts to 133 micrograms/kg in several Chinese salted and dried marine fish and in Greenland dried, unsalted fish preparations. NPYR and NPIP were also occasionally detected in several vegetables fermented in brine collected in Tunisia and China. The possible role of nitrosamines in the etiology of NPC is discussed.     

Volatile nitrosamine levels and genotoxicity of food samples from high-risk areas for nasopharyngeal carcinoma before and after nitrosation

Traditional life-style, especially food habits, infection by Epstein-Barr virus (EBV) and genetic factors, have been associated with an increased risk of nasopharyngeal carcinoma (NPC). N-Nitroso compounds and other carcinogens either present in food or formed endogenously, as well as food constituents that activate EBV, have been suspected as etiological factors in NPC pathogenesis. For their characterization preserved food items, frequently consumed in NPC endemic areas in Tunisia, South China and Greenland, were sampled and screened for the presence of mutagens and volatile nitrosamines before and after nitrosation. Aqueous extracts as well as 2 organic extracts of the samples were assayed for genotoxicity in 2 Salmonella typhimurium strains and the SOS chromotest. The same extracts had previously been analyzed for volatile nitrosamines and for EBV-activating substances in Raji cells. In our study, 13 out of 16 food samples showed a weak, directly-acting genotoxicity in the SOS chromotest in at least one of the extracts, but only one sample from Greenland was found to be weakly mutagenic in Salmonella TA 98. Chemical nitrosation for 9 out of 15 samples of aqueous food extracts increased the genotoxic effect in the SOS chromotest. Levels of volatile nitrosamines were also elevated for 12 out of 15 samples; highest levels of N-nitrosodimethylamine were found in hard salted and dried fish from China (1,200 micrograms/kg) and highest N-nitrosopyrrolidine levels in a Tunisian spice (3,840 micrograms/kg). In non-nitrosated aqueous food extracts, the level of volatile nitrosamines and genotoxic activities were not correlated with the EBV-inducing activity of the same samples. After chemical nitrosation, EBV-inducing activity was decreased or showed no change and was not correlated with increases in either the genotoxicity or the nitrosamine levels. Our results suggest that EBV-activating compounds belong to a different class of substances. However, there was an association between the changes in genotoxicity and nitrosamine levels due to nitrosation.     

Diffusion of nitrosamines through protective gloves

A simple experimental demonstration of the permeability of protective gloves to volatile nitrosamines has been carried out. Rubber and polyvinyl chloride (PVC) gloves were turned inside out and the fingers filled with dilute solutions of nitrosamines in hexane. The outer faces of the fingers were washed with water at various times and the nitrosamine content of these washings determined. From these measurements the proportions of the various nitrosamines passing through the gloves were calculated. For both types of gloves N-nitrosopyrrolidine (NPYR) permeated the fingers most rapidly and to the greatest extent. Only a relatively small amount of N-nitrosodibutylamine (NDBA) was transmitted. It was also established that the rubber gloves themselves did not contain any of the common volatile nitrosamines; N-nitrosomethylphenylamine and N-nitrosodiphenylamine were also absent.     

Combination experiments with very low doses of three genotoxic N-nitrosamines with similar organotropic carcinogenidty in rats

The study was designed to assess the syncarcinogenk activityof very low doses of N-nitrosodiethylamine (NDEA), N-nitrosopyrrolidine(NPYR) and N-nitrosodiethanolamine (NDE1A) in the liver of 1800male Sprague-Dawky rats. The N-nitrosamines were administeredthroughout the rats' lives individually and in combination atthree logarithmically spaced dose levels contained in drinkingwater. The dose levels in the individual dose-response experimentsranged from the lowest concentrations of previous experiments(NDEA, 0.1 mg/kg; NPYR, 0.4 mg/kg; NDE1A, 2.0 mg/kg) to dosages10 times lower and comprised a high, medium and low dose (escalationfactor: 3.16). The high dose of the combination contained thethree nitrosamine concentrations used as the medium doses ofthe individual nitrosamines. The medium combination dose resultedfrom the combined administration of the three lowest dosages,and the low combination dose consisted of three nitrosaminedosages which amounted to one-third of the low dosages respectively.Administration of these dosages was associated with a dose-dependentincidence of liver cancer: NDEA induced 45, 3.8 and 2.5%; NPYRcaused 21.3, 5 and 1.3%; NDE1A generated 7.5, 1.3 and 2.5%;and the combinations induced 16, 4.2 and 1.7%; respectively.Untreated controls showed 0.6% liver cancer incidence. Besidesthe liver, the gastrointestinal tract, the neurogenk tissue,the urinary tract and the hematopoietk and lymphatic tissuewere affected by tumor incidences increased over that of controls.There was, however, no well-defined dose dependency as withthe liver tumors. These results indicate dose dependency ofliver tumor formation even at very low exposure levels of theindividual agents. The carcinogenic effects of the hepatotropicN-nitrosamines summed up in combination. The observed additivitywas linear. Dose levels, which alone would presumably not havebeen carcinogenic, effected a significant cancer risk in combination.     

Chemoprevention of aflatoxin B1-induced genotoxicity and hepatic oxidative damage in rats by kolaviron, a natural bioflavonoid of Garcinia kola seeds.

The chemopreventive effects of kolaviron, a natural antioxidant bioflavonoid from the seeds of Garcinia kola, on aflatoxin B1 (AFB1)-induced genotoxicity and hepatic oxidative damage was investigated in rats. Kolaviron administered orally at a dose of 200 mg/kg once a day for the first 2 weeks and then 100 mg/kg twice a day for the last 4 weeks of AFB1 (2 mg/kg, single dose, intraperitoneal) treatment reduced the AFB1-increased activities of aspartate amino transferase (AST), alanine amino transferase (ALT) and gamma glutamyltransferase (gamma-GT) by 62%, 56% and 72% respectively. Malondialdehyde (MDA) formation and lipid hydroperoxide (LHP) accumulation were observed in the livers of AFB1-treated rats. Kolaviron significantly reduced the AFB1-induced MDA and LHP formation. Vitamins C and E were protective in reducing the increase in the activities of AST, ALT and gamma-GT as well as lipid peroxidation caused by AFB1 (P<0.01). Administration of rats with kolaviron alone resulted in significant elevation in the activities of glutathione S-transferase, uridyl glucuronosyl transferase and NADH:quinone oxidoreductase by 2.45-, 1.62- and 1.38-folds respectively. In addition, kolaviron attenuated the AFB1-mediated decrease in the activities of these enzymes (P<0.01). Pretreatment of rats with kolaviron, vitamins C and E alone did not exert genotoxicity assessed by the formation of micronucleated polychromatic erythrocytes (MNPCEs) (P>0.05). Co-treatment of rats intraperitoneally with kolaviron (500 mg/kg) 30 min before and 30 min after AFB1 (1 mg/kg) administration inhibited the induction of MNPCEs by AFB1 (P<0.001) after 72 h. While vitamin C was effective in reducing AFB1-induced MNPCEs formation, vitamin E did not elicit any antigenotoxic response. These results indicate kolaviron as effective chemopreventive agent against AFB1-induced genotoxicity and hepatic oxidative stress. Thus kolaviron may qualify for clinical trial in combating the menace of aflatoxicosis in endemic areas of aflatoxin contamination of foods.     

Comparative metabolism of N-nitrosopiperidine and N-nitrosopyrrolidine by rat liver and esophageal microsomes and cytochrome P450 2A3

N-nitrosopiperidine (NPIP) is a potent esophageal carcinogen in rats whereas structurally similar N-nitrosopyrrolidine (NPYR) induces liver, but not esophageal tumors. NPIP is a possible causative agent for human esophageal cancer. Our goal is to explain mechanistically these differing carcinogenic activities in the esophagus. We hypothesize that differences in metabolic activation of these nitrosamines could be one factor accounting for their differing carcinogenicity. alpha-Hydroxylation is the key metabolic activation pathway leading to nitrosamine-induced carcinogenesis. In this study, we examined the alpha-hydroxylation rates of [3,4-(3)H]NPIP and [3,4-(3)H]NPYR by male F344 rat esophageal and liver microsomes. The major alpha-hydroxylation products of NPIP and NPYR, 2-hydroxytetrahydro-2H-pyran (2-OH-THP) and 2-hydroxytetrahydrofuran (2-OH-THF), respectively, were monitored by high performance liquid chromatography with radioflow detection. NPIP or NPYR (4 microM) was incubated with varying concentrations of esophageal microsomes and co-factors. Microsomes converted NPIP to 2-OH-THP with a 40-fold higher velocity than NPYR to 2-OH-THF. Similar results were observed in studies with NPIP and NPYR at substrate concentrations between 4 and 100 micro M. Kinetics of NPIP alpha-hydroxylation were biphasic; K(M) values were 312 +/- 50 and 1600 +/- 312 microM. Expressed cytochrome P450 2A3, found in low levels in rat esophagus, was a good catalyst of NPIP alpha-hydroxylation (K(M) = 61.6 +/- 20.5 microM), but a poor catalyst of NPYR alpha-hydroxylation (K(m) = 1198 +/- 308 micro M). Cytochrome P450 2A3 may play a role in the preferential activation of NPIP observed in rat esophagus. Liver microsomes metabolized NPYR to 2-OH-THF (V(max)/K(M) = 3.23 pmol/min/mg/ microM) as efficiently as NPIP to 2-OH-THP (V(max)/K(M) = 3.80-4.61 pmol/min/mg/ microM). We conclude that rat esophageal microsomes activate NPIP but not NPYR whereas rat liver microsomes activate NPIP and NPYR. These results are consistent with previous findings that tissue-specific activation of nitrosamines contributes to tissue-specific tumor formation.     

Effects of long term dietary phenethyl isothiocyanate on the microsomal metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanol in F344 rats

Phenethyl isothiocyanate (PEITC), a cruciferous vegetable component, inhibits lung tumor induction by the tobacco specific nitrosamine, 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). To gain insight into the mechanism of PEITC lung tumor inhibition, we examined, in male F344 rats, the effects of dietary PEITC (3 micromol/g NIH-07 diet) in combination with NNK treatment (1.76 mg/kg, s.c., three times a week) for 4, 12 and 20 weeks on liver and lung microsomal metabolism of NNK and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a major metabolite of NNK and also a lung carcinogen. This was compared with rats fed NIH-07 diet, without PEITC, and treated with NNK alone or saline. The protocol was identical to that employed for inhibition of lung tumorigenesis by PEITC. We observed decreased rates of alpha- hydroxylation of NNK and NNAL in lung microsomes of 4-, 12- and 20-week PEITC + NNK treated rats compared with those treated with NNK or saline. NNK treatment alone also decreased lung alpha-methylene hydroxylation of NNK. Long-term NNK + PEITC administration did not significantly affect liver oxidative metabolism of NNK or NNAL, and did not affect the rate of glucuronidation of NNAL in liver microsomes when compared with rats treated with NNK or saline. Thus, PEITC selectively inhibited lung metabolic activation of NNK and NNAL. These results support the hypothesis that PEITC inhibits NNK-induced lung tumors by inhibiting metabolic activation of NNK in the lung. This study also demonstrated that PEITC inhibits lung alpha-hydroxylation of NNAL; this may play a role in PEITC inhibition of lung tumorigenesis by NNK.      

Formation of 7-(4-oxobutyl)guanine in hepatic DNA of rats treated with N-nitrosopyrrolidine.

We have reported previously the formation of two structurally distinct exocyclic guanine adducts (adducts 1 and 6) in liver DNA of F344 rats treated with N-nitrosopyrrolidine (NPYR). In this study, we detected and characterized a previously unidentified guanine adduct in liver DNA of NPYR-treated rats. The structure of this adduct was established as 7-(4-oxobutyl)guanine (adduct 2) by comparison with the synthetic standard and confirmed by NaBH4 reduction to 7-(4-hydroxybutyl)guanine. The level of adduct 2 in liver DNA of F344 rats treated with 450 mg/kg of NPYR by i.p. administration was 643 +/- 9 mumol/mol guanine, approximately one-third of the level of adduct 1. This study is the first to demonstrate the in vivo formation of a formylalkyl-substituted guanine adduct by a nitrosamine.     

Studies on the effect of feeding nitrite and secondary amines to Wistar rats.

1. When 1 000 mg/l sodium nitrite are added to drinking-water, nitrosamines are formed in the stomachs of Wistar rats at levels greater than the background only if the concentrations of added DMA or pyrrolidine exceed 1 000 mg/kg. Once this concentration is exceeded there is a rapid increase in nitrosamine formation up to 2 000 mg/kg added amine; however, for pyrrolidine, the rate of increase of NPy decreases when the dietary level of amine exceeds 2 000 mg/kg. This threshold level of 1 000 mg/kg amine is one which is rarely reached in normal human dietary patterns. 2. Due to the presence of this threshold it is unrealistic to extrapolate from high experimental dietary concentrations of secondary amines to those found in practice when considering nitrosamine formation in vivo. 3.The concentration of dietary amine has a greater influence on nitrosamine formation in the stomachs of rats than does the concentration of nitrite in drinking-water (up to 1 000 mg/l). This finding is in contradiction to the current kinetic theory of nitrosamine formation, in which formation is predicted to be proportional to the square of the nitrite concentration.     

Preliminary observations on amines and nitrosamines in non-normal human gastric contents.

Gastric contents from fasting humans were pooled and analysed for amines. Volatile amines present were dimethylamine, trimethylamine and histamine; non-volatile amines found were cadaverine, putrescine, ethanolamine and tryptamine. Gastric contents from 35 patients, some before and/or after gastric stimulation with histamine or pentagastrin, were analysed for nitrosamines. N-Nitrosodiethylamine (NDEA) (5-30 microgram/kg) was present in four samples, N-nitrosodimethylamine (NDMA) (2 microgram/kg) in two samples and N-nitrosopyrrolidine (NPYR) (6 microgram/kg) in one sample. pH, nitrite and nitrate determinations were made on some samples. Medical diagnosis of patients could not be correlated with the presence of nitrosamines in the gastric contents.     

Effects of butylated hydroxyanisole on the tumorigenicity and metabolism of N-nitrosodimethylamine and N-nitrosopyrrolidine in A/J mice

Female A/J mice were maintained on NIH-07 diet or on NIH-07 diet containing butylated hydroxyanisole (BHA, a mixture of 2- and 3-tert-butyl-4-hydroxyanisole), 5 mg/g, for 1 week prior to and during 10 weeks of treatment with N-nitrosodimethylamine (NDMA) or N-nitrosopyrrolidine (NPYR), administered in the drinking water. Twenty weeks after nitrosamine treatment ended, mice were sacrificed and lung adenomas were counted. BHA inhibited NDMA tumorigenesis but enhanced NPYR tumorigenesis. Treatment of A/J mice for three weeks with BHA (5 mg/g) added to semisynthetic diet increased whole lung microsomal alpha-hydroxylation of NDMA and NPYR, as measured by aldehyde production, and increased lung and hepatic glutathione-S-transferase activities. No evidence was found for formation of S-methylglutathione in incubations with NDMA and hepatic supernatants obtained from BHA treated or control mice. Four h after gavage of NDMA, levels of 7-methylguanine in the lung DNA of mice treated with BHA were higher than in the lung DNA of control mice, but levels of O6-methylguanine in the two groups were the same. The results of this study indicate that BHA treatment increases the microsomal metabolic alpha hydroxylation of both NDMA and NPYR, but has differential effects on their tumorigenic activities.     

A study of tobacco carcinogenesis. XLII. Bioassay in A/J mice of some structural analogues of tobacco-specific nitrosamines

The tumorigenic activities in A/J mouse lung of the tobacco-specific nitrosamines, N'-nitrosonornicotine (NNN), 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and several structural analogues were evaluated. The analogues were N-nitrosopyrrolidine (NPYR), 5'-carboxy-N'-nitrosonornicotine (CNNN), N-nitrosoproline (NPRO) and 1-(3-pyridyl)-2-buten-1-one (PBO). The results were as follows (dose in mumol per mouse/lung tumors per mouse): NNN (100/1.8 +/- 1.4); NPYR (100/3.9 +/- 1.5); CNNN (200/0.3 +/- 0.5); CNNN (100/0.5 +/- 0.6); NPRO (100/0.6 +/- 0.7); NNK (20/7.2 +/- 3.4); PBO (20/0.7 +/- 1.0); saline control (0.0.5 +/- 0.7). Several conclusions were drawn from this assay. NNK and NPYR were more tumorigenic than NNN. CNNN was non-tumorigenic and thus appears to have potential as a monitor for endogenous formation of tobacco-specific nitrosamines. The alpha,beta-unsaturated ketone PBO does not appear to be an ultimate tumorigen of NNK or NNN.     

Strong promoting activity of phenylethyl isothiocyanate and benzyl isothiocyanate on urinary bladder carcinogenesis in F344 male rats

Post-initiation effects of phenylethyl isothiocyanate (PEITC) and benzyl isothiocyanate (BITC) on hepatocarcinogenesis and urinary bladder carcinogenesis were examined in rats pretreated with diethylnitrosamine (DEN) and N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Groups of 21 rats received a single intraperitoneal injection of 200 mg/kg body weight of DEN. Starting 2 days thereafter, they were administered 0.05% BBN in the drinking water for 4 weeks. Three days after completion of the carcinogen treatment, they were placed on a diet containing PEITC or BITC at a dose of 0.1%, or a basal diet alone for 32 weeks and then killed for autopsy. Further groups of 6 rats each were similarly treated with PEITC, BITC or basal diet alone for 32 weeks without prior DEN and BBN exposure. In the liver, although the incidences of liver tumors were not significantly affected, the multiplicity of foci larger than 0.5 cm in diameter was slightly increased by PEITC. In the urinary bladder, the incidences of papillary or nodular (PN) hyperplasias and carcinomas were significantly elevated by PEITC or BITC after DEN and BBN initiation. In the groups without initiation, PN hyperplasia was found in all rats of both PEITC and BITC groups, along with papillomas and carcinomas in some animals. Tumors and PN hyperplasias in the groups treated with PEITC and BITC are characterized by downward growth. Our results thus showed PEITC and BITC to be strong promoters of urinary bladder carcinogenesis with some complete carcinogenic potential. Int. J. Cancer 77:773–777, 1998. © 1998 Wiley-Liss, Inc.     

Nitrosamines and their precursors in some Kazakh foodstuffs

Numerous samples of various foods, such as salt-dried and smoke-dried meats, fermented sausages, sour-milk products, fermented mare's and camel's milk, raw vegetables, rice, rock and table salts, local bread and daily food rations were analysed by GC-TEA and HPLC-TEA for the presence of volatile nitrosamines. Apart from the observation of NDMA (up to 0.7 microgram/kg) in three samples of Kazakh cheeses, the volatile nitrosamines were detected only in some meat and meat products. Salted air-dried meat contained NDMA in amounts up to 54 micrograms/kg. NPYR (12 micrograms/kg) was found in one sample of nitrite-cured sausage. The highest content of NPIP (7 micrograms/kg) was detected in fermented, home-made sausage. It is clear that the only significant contribution of these dialkyl and heterocyclic nitrosamines to the diet is from stored, salt-dried meat and nitrite-cured meat products. Analyses of nitrate and nitrite in various local products are reported. The validity of our assumption that the high incidence of oesophageal cancer can be lowered by realistic improvements in the processing and storage of meat and meat products requires further evaluation.     

Metabolic activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone as measured by DNA alkylation in vitro and its inhibition by isothiocyanates

The bioactivation of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), by microsomes from target organs was studied with an in vitro microsome-mediated DNA alkylation system. Mouse lung, rat lung, and rat nasal microsomes catalyzed a time- and protein-dependent DNA methylation by [methyl-3H]NNK with activities of 4.11, 0.95, and 137.4 pmol/mg DNA/mg protein/h, respectively. The DNA methylation of NNK catalyzed by all three microsomal systems was inhibited by cytochrome P-450 inhibitors, such as carbon monoxide and metyrapone, but not by the cyclooxygenase inhibitor, aspirin, or by prolonged preincubation in the absence of NADPH. The possible involvement of specific P450 isozymes was assessed by specific inhibitory antibodies. An anti-P450IIB1&2 antibody significantly inhibited the DNA methylation by 45 and 32% in mouse lung and rat lung, respectively, whereas anti-P450IA1 and anti-P450IIE1 antibodies failed to show significant inhibition. All antibodies showed no inhibition in rat nasal microsomes. Glutathione inhibited the DNA methylation in a concentration-dependent manner in all three microsomal systems. Phenethyl isothiocyanate (PEITC), at doses of 0.25 and 1.00 mmol/kg body weight, was given intragastrically 2 h before sacrifice to mice and 24 h before sacrifice to rats, respectively; both mouse and rat lung microsomal activities were inhibited by about 40 and 90% by the low- and high-dose PEITC treatments, respectively. The rat nasal microsomes were only inhibited by the high-dose PEITC treatment by about 40%. PEITC, 4-phenylbutyl isothiocyanate, and 6-phenylhexyl isothiocyanate all inhibited the microsome-mediated DNA methylation of NNK in vitro, with 4-phenylbutyl isothiocyanate and 6-phenylhexyl isothiocyanate being more potent than PEITC and the mouse lung microsomes more sensitive than the rat lung and nasal microsomes. All three microsomal systems were shown to catalyze the in vitro DNA pyridyloxobutylation by [5-3H]NNK. On an equal protein basis, the rat nasal microsomes were much more active in catalyzing the DNA pyridyloxobutylation.     

Structure-activity relationships for inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone lung tumorigenesis by arylalkyl isothiocyanates in A/J mice

Phenethyl isothiocyanate (PEITC), 3-phenylpropyl isothiocyanate (PPITC), 4-phenylbutyl isothiocyanate (PBITC), and the newly synthesized 5-phenylpentyl isothiocyanate (PPeITC), 6-phenylhexyl isothiocyanate (PHITC), and 4-(3-pyridyl)butyl isothiocyanate (PyBITC) were tested for their abilities to inhibit tumorigenicity and DNA methylation induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the lungs of A/J mice. Mice were administered isothiocyanates by gavage for 4 consecutive days at doses of 5, 1, or 0.2 mumol/day prior to administration of 10 mumol of NNK by i.p. injection. Mice were sacrificed 16 weeks after NNK administration and pulmonary adenomas were quantitated, PEITC effectively inhibited NNK-induced lung tumors at a dose of 5 mumol/day but was not inhibitory at doses of 1 or 0.2 mumol/day. PPITC, PBITC, PPeITC, and PHITC were all considerably more potent inhibitors of NNK lung tumorigenesis than PEITC. While virtually no differences in inhibitory activity could be ascertained for PPITC, PBITC, and PPeITC, PHITC appeared to be the most potent tumor inhibitor of all of the compounds. At a dose of 0.2 mumol/day, PHITC pretreatment reduced tumor multiplicity by 85%. PyBITC, an analogue of both NNK and PBITC, was ineffective as an inhibitor. Using the same protocol, the compounds were found to have qualitatively similar inhibitory effects on NNK-induced DNA methylation when administered at 1 mumol/day. These results extend our previous findings that increased alkyl chain length enhances the inhibitory activity of an arylalkyl isothiocyanate toward NNK lung tumorigenesis and demonstrate the exceptional chemopreventive potentials of two new isothiocyanates, PPeITC and PHITC.     

Effects of aromatic isothiocyanates on tumorigenicity, O6-methylguanine formation, and metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in A/J mouse lung

Phenethyl isothiocyanate (PEITC), benzyl isothiocyanate (BITC), and phenyl isothiocyanate (PITC) were tested for their abilities to inhibit lung tumorigenesis and O6-methylguanine formation in lung DNA induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice. Pretreatment with PEITC for 4 consecutive days at daily doses of 5 or 25 mumol inhibited tumor multiplicity induced by a single 10-mumol dose of NNK by approximately 70% or 97%, respectively. The 25-mumol daily dose of PEITC also reduced the percentage of animals that developed tumors by 70%. In contrast, both BITC and PITC failed to significantly reduce tumor multiplicity or the percentages of mice that developed tumors. Using an identical dosing regimen, parallel results were observed in the effects of these isothiocyanates on O6-methylguanine formation in the lung, in which PEITC at either dose resulted in considerable inhibition at 2 or 6 h after NNK administration, while BITC or PITC had little effect. PEITC was further tested for its ability to inhibit lung microsomal metabolism of NNK. A single administration of PEITC (5 or 25 mumol) resulted in 90% inhibition of NNK metabolism. These results in conjunction with recent results obtained using F344 rats firmly establish PEITC as an effective inhibitor of NNK lung tumorigenesis and suggest that the basis of this inhibition is the reduction of DNA adduct formation caused by the inhibition of enzymes responsible for NNK activation.