Chicken egg fetal liver DNA and histopathologic effects of structurally diverse carcinogens and non-carcinogens.

Chicken egg fetal livers were evaluated for histopathological changes produced by four genotoxic hepatocarcinogens: 2-acetylaminofluorene (AAF), aflatoxin B1 (AFB1), benzo[a]pyrene (BaP), diethylnitrosamine (DEN); four structurally related non- or weakly- carcinogenic comparators: fluorene (FLU), aflatoxin B2 (AFB2), benzo[e]pyrene (BeP), N-nitrosodiethanolamine (NDELA); two epigenetic hepatocarcinogens: clofibric acid (CFA), phenobarbital (PB); and the non-carcinogen, D-mannitol (MAN). CFA, PB and MAN were also assessed for formation of DNA adducts using the 32P nucleotide postlabeling (NPL) assay and for DNA breaks using the comet assay. CFA was also assessed in enhanced comet assay for oxidative DNA damage induction. Eggs were dosed on days 9- 11 of incubation. For genotoxicity evaluation, livers were collected 3h after the last dose. Liver qualitative histopathology assessment was performed on days 12 and 18 of incubation. CFA was negative for DNA adducts but yielded clear evidence of DNA breaks due to oxidative stress. PB and MAN produced no DNA adducts or breaks. Liver to body weight ratios were not affected in most groups, but were decreased in DEN groups, and increased after PB dosing. Livers from control groups, FLU, AFB2, BeP, NDELA, CFA, and MAN groups, displayed a typical hepatocellular trabecular pattern at both time points. In contrast, the four genotoxic carcinogens induced time- and dose- related interference with fetal liver cell processes of proliferation, migration and differentiation, leading to hepatocellular and cholangiocellular pleomorphic dysplasia and re-(de-) differentiation with distortion of the trabecular pattern. In addition, dosing with the high dose of DEN produced gallbladder agenesis. PB induced hepatocellular hypertrophy, interference with migration, expressed as distortion of the trabecular pattern, and a moderate cholangiocellular dysplasia. In summary, histopathological analysis of chicken fetal livers revealed developmental anomalies, as well as genotoxicity-induced and, in the case of PB, adaptive morphological changes. Thus, the model provides histopathological outcomes of molecular effects.

Inter-laboratory comparison of turkey in ovo carcinogenicity assessment (IOCA) of hepatocarcinogens.

In three independent laboratories carcinogens (diethylnitrosamine, DEN, 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone, NNK) and non-carcinogens (N-nitrosoproline, nicotine) were evaluated in turkey eggs for in ovo carcinogenicity assessment (IOCA). Compounds were injected into aseptic fertilized eggs. After incubation for 24 days, foci of altered hepatocytes (FAH), some with a pseudoglandular structure and/or signs of compression of the surrounding tissue were observed in the fetal liver. All laboratories were able to distinguish unequivocally the hepatocarcinogen-exposed groups from those exposed to non-carcinogens or the vehicle controls, based on the pre-specified evaluation parameters: tumor-like lesions, pseudoglandular areas and FAH. In addition to focal changes, only the carcinogens induced hepatocellular karyomegaly. Lower doses of the carcinogens, which did not induce FAH, were sufficient to induce hepatocellular karyomegaly. After exposure to 4 mg DEN, gall bladder agenesis was observed in all fetuses. The IOCA may be a valuable tool for early investigative studies on carcinogenicity and since it does not use rodents may complement chronic rat or mouse bioassays. Test substances that are positive in both rodents and fertilized turkey eggs are most probably trans-species carcinogens with particular significance for humans. The good concordance observed among the three laboratories demonstrates that the IOCA is a reliable and robust method.

Chemical profile of two types of oral snuff tobacco.

The chemical-analytical profile of two US brands of oral moist snuff was determined. These two brands were bought in five geographical locations (NY, MA, CO, CA and KY in the US). They were mixed thoroughly to yield representative samples. Brand A had a pH of 5.84 and nicotine content of 0.42%, while brand B had a pH of 7.99 and nicotine content of 2.73%. At pH 5.84, only 1% of the nicotine is present as a free base while 59% of nicotine is present in unprotonated form at pH 7.99. It is the unprotonated form of nicotine that is most readily absorbed through the mucous membrane in the oral cavity. Snuff A contained also significantly lower levels of moisture, nitrate, nitrite and tobacco-specific nitrosamines than snuff B. The University of Kentucky reference snuff 1S3 was analyzed as an external control sample. These two snuff brands are currently being assayed with rats in a short-term and in long-term bioassays to test the concept that the tobacco-specific N-nitrosamines are major contributors to the carcinogenic activity of oral snuff.

Exposure to N-nitroso compounds in a population of high liver cancer regions in Thailand: volatile nitrosamine (VNA) levels in Thai food.

The recent case-control studies in Thailand indicate that a high incidence of liver cancer in Thailand has not been associated with common risk factors such as hepatitis B infection, aflatoxin intake and alcohol consumption. While the infestation by the liver fluke Opisthorchis viverrini (OV) accounted for the high risk in north-east Thailand, there was no such exposure in the other regions of the country where the incidence of liver cancer is also high. Case-control studies suggest that exposure to exogenous and possibly endogenous nitrosamines in food or tobacco in betel nut and cigarettes may play a role in the development of hepatocellular carcinoma (HCC), while OV infestation and chemical interaction of nitrosamines may also be aetiological factors in the development of cholangiocarcinoma (CCA). Over 1800 samples of fresh and preserved food were systematically collected and tested between 1988 and 1996. All the food items identified by anthropological studies to be consumed frequently in four major regions of Thailand were analysed for volatile nitrosamines using gas chromatography combined with a thermal energy analyser. Relatively high levels of N-nitrosodimethylamine (NDMA), N-nitrosopiperidine (NPIP) and N-nitrosopyrrolidine (NPYR) were detected in fermented fish ("Plasalid"). NDMA was also detected at levels ranging from trace amounts to 66.5 microg/kg in several salted and dried fish ("Larb-pla" and "Pla-siu"). NDMA and NPYR were frequently detected in several vegetables, particularly fermented beans ("Tau-chiau") at levels ranging between 1 and 95.1 microg/kg and 0-146 microg/kg, respectively. The possible role of nitrosamines in Thai food in the aetiology of liver cancer (HCC, CCA) is discussed.

Volatile nitrosamines and tobacco-specific nitrosamines in the smoke of Thai cigarettes: a risk factor for lung cancer and a suspected risk factor for liver cancer in Thailand.

In Thailand, smoking of commercial cigarettes and of handmade cigarettes has drastically increased in recent decades. Cancer of the lung and of the upper aero-digestive tract have also increased in Thailand as they have in many other countries. It is our working hypothesis that the increase of primary cancer of the liver, especially of cholangiocarcinoma in the north-eastern provinces of Thailand is associated with the use of tobacco in men infested with the liver fluke Opisthorchis viverrini (OV). Bioassays have shown that volatile nitrosamines and tobacco-specific nitrosamines induce cholangiocarcinoma in laboratory animals and that the hepatocarcinogenic action of nitrosodimethylamine in hamsters is significantly increased by infestation with the liver fluke OV. The endogenous formation of nitrosamines is significantly increased by OV infestation. This report presents analytical data on the concentration of volatile nitrosamines and tobacco-specific nitrosamines in mainstream smoke of nine leading brands of commercially produced Thai cigarettes which represent approximately 85% of the market share in Thailand. Observed ranges (ng/cigarette) were 8.5-31.9 for nitrosodimethylamine, 8.8-49.6 for nitrosopyrrolidine and 4.2-18.9 for nitrosodi-n-butylamine. These values are exceptionally high compared with the smoke of light and blended cigarettes from North America and Western Europe. Among the tobacco-specific nitrosamines, the range was 28-730 for nitrosonornicotine and 16-370 for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. There was a correlation between volatile and tobacco-specific nitrosamines, and tar and nicotine deliveries in the mainstream smoke. The analytical data are in line with the rate for lung cancer and support our working hypothesis that nitrosamines, and especially the tobacco-specific nitrosamines, are associated with the increased risk for primary liver cancer among those Thai people who smoke cigarettes and also carry OV infestation.

Short- and intermediate-term carcinogenicity testing--a review. Part 2: available experimental models.

Numerous experimental protocols for short- and intermediate-term carcinogenicity assays have been available for many years. This paper surveys various of these test systems in rodents, fish species, non-vertebrates and avian embryos in ovo. The mouse skin tumour assay and the rat liver foci assay were used to introduce the basic concepts of short- and intermediate-term carcinogenicity testing in the previous part of the review. The focus of this second part of the review is on rodent assays for carcinogenicity testing in the lung, kidney, urinary bladder, pancreas, stomach, oral cavity, small intestine, colon, and on the possibility to combine several target organs in multi-organ models. The potential use of various fish species, non-vertebrates and hatching eggs for carcinogenicity testing is outlined and the advantages and limitations are discussed. This review also presents the problem of validation of any carcinogenicity test system and proposes a strategy for contemporary safety assessment of chemicals with regard to the detection and evaluation of carcinogenicity.

The in ovo carcinogenicity assay (IOCA): a review of an experimental approach for research on carcinogenesis and carcinogenicity testing.

Up to now, the carcinogenicity of a substance, i.e., its cancer-causing effect, has been usually determined with the help of extensive animal testing. The in ovo carcinogenicity assay (IOCA) has been suggested as a rapid and inexpensive, non-animal, method for carcinogenicity testing and for experimental studies on mechanisms of carcinogenesis. The substance to be tested is injected into a fertilized ovum. No later than four days before the hatching date, the embryos are released, and the liver is removed for identifying the effect of hepatocarcinogens. Histological, cytological and molecular biological alterations of the embryonic liver have been induced with a variety of chemical carcinogens. After sufficiently high doses of hepatocarcinogens, tubular structures predominate in the liver and replace the normal trabecular pattern. The cell and nuclear size of the hepatocytes in embryonic liver is severely increased after exposure to chemical carcinogens over a wide dose range including doses that fail to elicit cytotoxicity in the embryonic liver.

Assessment of chronic toxicity and carcinogenicity in rats of Wingstay 100, a rubber antioxidant/antiozonant.

The chronic toxicity and carcinogenicity of Wingstay 100 (W 100), a rubber antioxidant/antiozonant, were studied in Fischer 344 (F 344) rats in two chronic studies. Earlier genetic studies indicated that the product had weak activity in a bacterial mutation assay, but lacked activity in chromosomal aberration assays. In an one year study, both genders of F 344 rats were exposed to 53, 310 or 1900 ppm in NIH-07 diet for 52 weeks, and sacrifices were made at 38, 52 and 64 weeks. No test substance related deaths occurred, although the high dose of 1900 ppm caused a decrease in body weight gain and food consumption in both genders. Red blood cell mean corpuscular volume was significantly increased at 38 weeks, accompanied by a significant decrease in mean corpuscular hemoglobin concentration. At 52 weeks, the red blood cell count and hemoglobin values were also significantly decreased in high dose animals of both genders. Total bilirubin and cholesterol were increased in high dose animals of 38 and 52-week sacrifices. During the 3 month recovery, hematology parameters, bilirubin and cholesterol returned to control values. Total protein was reduced in high dose animals of both genders, throughout the entire exposure and recovery periods. W 100 also produced increases in relative liver, spleen, heart and kidney weights in high dose animals. Both genders of all W 100 groups exhibited significant increases in urothelial cell proliferation (measured by PCNA) and adaptive hyperplasia. No regenerative hyperplasia, preneoplasia, or neoplasia were present. There was microscopic evidence of extramedullary erythropoiesis in the spleen and liver of high dose animals in both genders, otherwise no other pertinent microscopic finding was evident. In parallel, an accelerated bioassay (ABA) was conducted, which is a mechanistic initiation/promotion carcinogenicity study designed to assess tumor induction and promotion potential of a test substance in major organs of carcinogenesis. The present study was conducted in male F 344 rats for 38 weeks. The target sites chosen for the ABA were liver and urinary bladder and the dose for W 100 was 1900 ppm previously established to be a toxic dose. The liver tumor initiator was diethylnitrosamine (DEN), and the urinary bladder initiator was N-butyl N-(4-hydroxybutyl) nitrosamine (BBN). The initiators were administered during the first 14 weeks followed by the promoters. The promoters, phenobarbital (PB) for the liver and nitrilotriacetate (NTA) for the urinary bladder, were administered during the last 24 weeks of the study after the test substance. The study had 11 test groups including a negative control. The critical comparisons for initiating activity were conducted between groups 3 (PB) and 6 (W 100 + PB) for the liver and groups 8 (NTA) and 11 (W 100 + NTA) for the urinary bladder. The critical comparisons for promoting activity were conducted between groups 2 (DEN) and 5 (DEN + W 100) for the liver and groups 7 (BBN) and 10 (BBN + W 100) for the urinary bladder. There were 26 and 38-week sacrifices. In this study, most body weight reductions were due to DEN. At 26 weeks, significant increases in liver weights were present in all PB-exposed groups. Significant increases in renal weights occurred in all NTA, BBN and DEN groups. A similar organ weight pattern was present at 38 weeks. At 26 weeks, there were hepatocellular (33%) and urothelial (67%) tumors present in positive control groups (DEN/DEN + PB/BBN/BBN + NTA). In contrast, in the DEN + W 100 (5) and the BBN + W 100 (10) groups no tumors were present indicating absence of promotion. In addition, no tumors were present in groups 6 (W 100 + PB) or 11 (W 100 + NTA) indicating absence of initiation. At 38 weeks, the incidences of hepatocellular adenomas and carcinomas in positive control group (DEN) was 44%. The incidence of urothelial adenomas and carcinomas was 67% in group 7 (BBN). In contrast, groups 5 (DEN + W 100) or group 10 (BBN + W 100) had

Assessment of major carcinogenic tobacco-specific N-nitrosamines in Thai cigarettes.

This report presents new findings on the content of cancer-causing tobacco-specific N-nitrosamines (TSNA) in mainstream smoke of nine brands of commercially produced Thai cigarettes, representing about 85% of market share in Thailand. Also tested were two major and popular brands of U.S. cigarettes sold in Thailand, representing about 10% of market share. The cigarettes included filter and nonfilter cigarettes with high, moderate, and low tar and nicotine yields. The observed range for N-nitrosonornicotine (NNN) was from 28 to 730 ng/cigarette and for 4-(methylnitrosamino)-1-(3-pyridyl-1-butanone (NNK) from 16 to 369 ng/cigarette. The relatively highest TSNA values were obtained in filter and nonfilter cigarettes with high tar (22.3-28.1 mg/cigarette) and high nicotine (1.78-2.42 mg/cigarette) deliveries. The results demonstrated that there is a correlation between TSNA and tar and nicotine deliveries in mainstream smoke. The TSNA deliveries, along with the tar and nicotine levels in mainstream smoke depended on the tobacco composition. According to these results, the tar levels alone, while significant, are not a sufficient measure for the biological activity and the carcinogenic potential of cigarettes in Thailand. Consumption of tobacco products nearly quadrupled over the last three decades (1966-1995) in Thailand. Lung cancer is the leading malignancy for men and a common malignancy for women in Thailand. This report provides information that may prove helpful in evaluating the TSNA-carcinogens burden on smokers. Our goal is to offer the scientific basis for voluntary and/or government-regulated reduction of the smoke yields of TSNA in tobacco products in Thailand and in other countries.

Formation and analysis of tobacco-specific N-nitrosamines.

Chemical-analytical studies during the past 4 years led to several new observations on the formation of tobacco-specific N-nitrosamines (TSNA) and their occurrence in smokeless tobacco, mainstream smoke (MS), and sidestream smoke (SS) of American and foreign cigarettes. When snuff was extracted by means of supercritical fluid extraction with carbon dioxide containing 10% methanol, analysis of this material confirmed that the extraction with organic solvents had been partially incomplete. Epidemiological studies in the northern Sudan showed a high risk for oral cancer for users of toombak, a home-made oral snuff. Toombak contains 100-fold higher levels of TSNA than commercial snuff in the U.S. and Sweden. The TSNA content in the saliva of toombak dippers is at least ten times higher than that reported in the saliva of dippers of commercial snuff. Biomarker studies have shown corresponding high levels of hemoglobin adducts with metabolites of NNN and NNK as well as for urinary metabolites of NNK. These data supported the epidemiological findings. The analyses of MS of U.S. and foreign cigarettes smoked under FTC conditions revealed comparable data for the smoke of nonfilter cigarettes and filter cigarettes except in the case of low- and ultralow-yield cigarettes, which showed reduced TSNA yields. The MS of cigarettes made from Burley or dark tobacco is exceptionally high in TSNA, primarily because of the high nitrate content of those tobacco types. Taking puffs of larger volume and drawing puffs more frequently, practices observed among most smokers of cigarettes with low nicotine yield, results in high TSNA values in the MS. The formation of the lung carcinogen NNK is favored during the smoldering of cigarettes, between puffs, when SS is generated. Consequently, in most samples from indoor air polluted with environmental tobacco smoke (ETS), the highest concentration of an individual TSNA is that of NNK. When nonsmokers had remained for up to 2 h in a test laboratory with high ETS pollution, they excreted measurable amounts of NNK metabolites in the urine, indicative of the uptake of TSNA.

Combination chemotherapy for advanced lung cancer.

Lung cancer is now a major public health problem in Thailand. This study was undertaken to gain some preliminary data regarding the potential effectiveness in treating advanced non-small cell lung carcinoma (NSCLC) using an ifosfamide combination therapy IA(E)P. A clinical study was made of all 50 patients (Thais) with histologically proven, advanced NSCLC admitted to the University of Siriraj Hospital between 1985 and 1987 and followed up until February 1992. Survival was calculated for responders and non-responders as distinct groups, and for the different histological tumors among the responders. There were 22 cases of adenocarcinoma, 13 large cell carcinoma, and 15 squamous carcinoma. Twenty-seven out of 50 (54%) responded to treatment. The median survival of the response group was 17 months, compared with 5.5 months in the nonresponse group. The longest survival period was seen in patients with large cell carcinoma. The results suggest that moderate success might be expected in selected patients using the IA(E)P. Further work should be undertaken in developing countries using controlled clinical trials to more fully determine the efficacy of IA(E)P in treating NSCLC.

Endogenous formation of N-nitrosomorpholine in mice from 15NO2 by inhalation and morpholine by gavage.

Male CD-1 mice were exposed to an nominal concentration of 20 p.p.m. of 15N-nitrogen dioxide (15NO2) for 6 h/day for 4 days and for 2 h on the day 5, and to 1 g morpholine/kg body wt by gavage daily for five consecutive days. N-Nitrosomorpholine (NMOR) was found in whole mice, stomachs, skins with hair, and remains. The sum of individual tissue concentrations measured separately was 3421 ng/tissue, where the average skin weighed 4.3 g, the average stomach weighed 1.0 g and the average remains weighed 22.2 g. The average whole mouse weighed 27.7 g and contained a total of 3903 ng of NMOR. The concentration of NMOR was highest in the skin, next highest in the stomach, and lowest in the remains. However, the total quantity of NMOR per tissue, while highest in the skin (83%), was next highest in the remains (14.8%) and lowest in the stomach (2.2%). GC-MS analysis served to distinguish between the NMOR of 15NO2 origin and that of other origin. All of the NMOR in the whole mouse homogenates was identified as 15NMOR. In the stomach 73% was identified as 14NMOR, representing 1.6% of the total NMOR in the mouse, and 27% as 15NMOR, representing 0.6% of the total NMOR in the mouse. N-Nitrosamine formation in vivo is discussed as a possibly ongoing mammalian process.

Low ignition propensity cigarettes: smoke analysis for carcinogens and testing for mutagenic activity of the smoke particulate matter.

In 1990, more than 20% of the almost 5200 deaths from fires in the United States occurred in fires from cigarettes. This fact led to the investigation of cigarettes with low ignition propensity. 32 experimental cigarettes differing in tobacco, cigarette circumference and in cigarette paper were tested for ignition propensity. This communication reports the results of comparative analyses of smoke from two low-propensity experimental cigarettes, A and B, which differed only in the porosity and treatment of the paper, and of the smoke from a reference cigarette C and from two leading US commercial cigarettes D and E. Model cigarette A delivered higher smoke yields of total particulate matter (TPM), nicotine, CO and benzo[a]pyrene than the other four cigarettes, but had lower smoke yields of the carcinogenic, volatile, and tobacco-specific N-nitrosamines than cigarettes C, D and E. The TPM of cigarette A was less active as a frameshift mutagen in tests with Salmonella strain TA98 than was the TPM of cigarettes C, D and E. TPM of cigarette A was also less active as a frameshift mutagen in tester strain TA1538 than was the TPM of reference cigarette C. However, when the mutagenic potencies of the particulate matters were compared on a cigarette-to-cigarette basis, there were no significant differences between the TPM of the individual cigarettes. Replacing the conventional cigarette filter with a perforated filter may significantly reduce the smoke yield of cigarette A. If this can be achieved without changing the ignition propensity, detailed chemical-analytical analyses and in vitro and in vivo assays for toxicity, ciliatoxicity, mutagenic activity and carcinogenicity need to be obtained for the smoke of such a prototype cigarette.

Histology and smoking history of lung cancer cases and implications for prevention in Thailand.

BACKGROUND: Lung cancer is now a major public health problem in Thailand. This descriptive study looked at the issue of whether tumor histology varied with smoking status among lung cancer patients in Bangkok, Thailand. METHODS: A retrospective descriptive study was made of the 1,600 Thai patients with histologically proven lung cancer admitted to University of Siriraj Hospital between 1967 and 1991. Cigarette smoking histories were obtained, and histologies were classified and related to tobacco consumption. RESULTS: Overall, 78% were smokers, 72% being heavy smokers. The male to female ratio was 13:1 for smokers and 0.4:1 for nonsmokers. There were 29% squamous cell, 29% adenocarcinoma, 24% large cell, and 13% small cell carcinomas. Squamous cell carcinoma was significantly more frequent among cases with a history of smoking Thai cigarettes, which are known (from other studies) to be high in tar and nicotine, than among nonsmoking cases. Among the 350 nonsmokers, 252 of whom were female, adenocarcinoma was the most common (58%). CONCLUSIONS: The results suggest that Thai smokers can reduce their risk for lung cancer by quitting smoking, or by substituting lower tar brands. Antismoking programs should therefore be a public health priority. Given the prevalence of adenocarcinoma among nonsmokers, further work should be done to identify environmental causes.

Tobacco-specific N-nitrosamines and Areca-derived N-nitrosamines: chemistry, biochemistry, carcinogenicity, and relevance to humans.

Nicotine and the minor tobacco alkaloids give rise to tobacco-specific N-nitrosamines (TSNA) during tobacco processing and during smoking. Chemical-analytical studies led to the identification of seven TSNA in smokeless tobacco (< or = 25 micrograms/g) and in mainstream smoke of cigarettes (1.3 micrograms TSNA/cigarette). Indoor air polluted by tobacco smoke may contain up to 24 pg/L of TSNA. In mice, rats, and hamsters, three TSNA, N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), are powerful carcinogens; two TSNA are moderately active as carcinogens; and two TSNA appear not to be carcinogenic. The TSNA are procarcinogens, agents that require metabolic activation. The active forms of the carcinogenic TSNA react with cellular components, including DNA, and with hemoglobin (Hb). The Hb adducts in chewers and smokers serve as biomarkers for the uptake and metabolic activation of carcinogenic TSNA and the urinary excretion of NNAL as free alcohol and as glucuronide for the uptake of TSNA. The review presents evidence that strongly supports the concept that TSNA contribute to the increased risk for cancer of the upper digestive tract in tobacco chewers and for the increased risk of lung cancer, especially pulmonary adenocarcinoma, in smokers. The high incidence of cancer of the upper digestive tract especially among men on the Indian subcontinent has been causally associated with chewing of betel quid mixed with tobacco. In addition to the TSNA, the betel quid chewers are exposed to four N-nitrosamines that are formed during chewing from the Areca alkaloids, two of these N-nitrosamines are carcinogens. The article also reviews approaches toward the reduction of the carcinogenic potency of smokeless tobacco, betel quid-tobacco mixtures, and cigarette smoke. Although the safest way to reduce the risk for tobacco-related cancers is to refrain from chewing and smoking, modifications of smokeless tobacco and of cigarettes are indicated to lead to less toxic products. Another more recent approach for reducing the carcinogenic effect of tobacco products is the application of chemopreventive agents, primarily of micronutrients. Future aspects in tobacco carcinogenesis, especially as it relates to TSNA, are expected in the field of molecular biochemistry and in biomarker studies, with the goal of identifying those tobacco and betel quid chewers and tobacco smokers who are at especially high risk for cancer.

A tobacco-specific lung carcinogen in the urine of men exposed to cigarette smoke.

BACKGROUND: Environmental tobacco smoke has been classified by the Environmental Protection Agency as a carcinogen causally associated with lung cancer in adults, but there have been no reports of lung carcinogens or their metabolites in the body fluids or tissues of nonsmokers exposed to environmental tobacco smoke. METHODS: Five male nonsmokers were exposed to sidestream cigarette smoke generated by machine smoking of reference cigarettes for 180 minutes on each of two days, six months apart. Sidestream smoke is the smoke that originates from the smoldering end of a cigarette between puffs. Twenty-four-hour urine samples were collected before and after exposure. The urine samples were analyzed for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronide, which are metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a powerful lung carcinogen in rodents. NNAL is also a lung carcinogen in rodents. RESULTS: The urinary excretion of the metabolites increased after exposure to sidestream smoke in all the men. The mean (+/- SD) amount of NNAL and NNAL glucuronide was significantly higher after exposure than at base line (33.9 +/- 20.0 vs. 8.4 +/- 11.2 ng per 24 hours [127 +/- 74 vs. 31 +/- 41 pmol per day], P < 0.001) and was correlated with urinary cotinine excretion (r = 0.89, P < 0.001). The nicotine concentrations in the air to which the men were exposed were comparable to those in a heavily smoke-polluted bar. CONCLUSIONS: Nonsmokers exposed to sidestream cigarette smoke take up and metabolize a lung carcinogen, which provides experimental support for the proposal that environmental tobacco smoke can cause lung cancer.

The need for regulation of carcinogenic N-nitrosamines in oral snuff.

Oral snuff is carcinogenic to humans and laboratory animals. The major carcinogenic agents in snuff are the N-nitrosamines, especially the tobacco-specific N-nitrosamines. During the past decade, a gradual reduction of the levels of carcinogenic N-nitrosamines was observed in the leading snuff brands in the USA and in Sweden. However, in 1990 a newly introduced snuff brand in the USA contained the highest concentration of carcinogenic N-nitrosamines ever to be determined in a commercial tobacco product. The elevated pH and relatively high levels of nitrite in this snuff favoured the formation of N-nitrosamines. 2 yr after the product first appeared, it was replaced by a new preparation of snuff under the same brand name, and, according to chemical analyses, this material would be expected to have about the same carcinogenic potential as the leading snuff products. The interdependence of the formulation and manner of preparation of snuff products with their carcinogenic potential emphasizes the need for regulation and control of the harmful substances in smokeless tobacco, especially in view of the trend of increasing consumption of snuff.

Analysis of tobacco-specific N-nitrosamines in indoor air.

A method was developed and applied for the assessment of tobacco-specific N-nitrosamines (TSNA) in indoor air polluted with tobacco smoke. Air samples were collected on Cambridge filters treated with 0.01 M potassium bisulfate, extracted with dichloromethane and enriched by column chromatography. The fraction containing the TSNA was concentrated and placed on a thermal desorption cartridge packed with Tenax GR. The sample was thermally desorbed and analyzed by capillary GC using a thermal energy analyzer. When the method was applied in a test laboratory in which one, two and four cigarettes were smoked during 30 min, linearity was observed. Field studies included sampling in bars, restaurants and trains. The concentration of N'-nitrosonornicotine (NNN) ranged from not detected to 23 pg/l, that of N'-nitrosoanatabine ranged from not detected to 9 pg/l, while 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was detected in concentrations ranging from 1 to 29 pg/l. This means an exposure to NNN and NNK of 0.1-0.3 cigarette equivalents. Thus, non-smokers can be exposed to highly carcinogenic TSNA.

A study of tobacco carcinogenesis. XLVII. Bioassays of vinylpyridines for genotoxicity and for tumorigenicity in A/J mice.

3-Vinylpyridine is formed from nicotine during the smoking of tobacco products. Consequently it is found in mainstream and side-stream smoke of cigarettes and cigars and in environmental tobacco smoke. In this study, 3-vinylpyridine and its isomers 2- and 4-vinylpyridine as well as styrene (vinylbenzene) were bioassayed for mutagenicity in Salmonella typhimurium strains TA 1535, TA 1538, TA 98 and TA 100 and for their genotoxicity in the rat hepatocyte DNA-repair test. In both in vitro assays-all three vinylpyridines and styrene were inactive. In a test for tumorigenicity in which the test compounds were injected intraperitoneally into A/J mice (total dose 200 mumol/animal) there was no significant incidence of lung adenoma nor of any other type of tumors.