Antioxidant, hepatoprotective and hypolipidemic effects of methanolic root extract of Cassia singueana in rats following acute and chronic carbon tetrachloride intoxication.

OBJECTIVE: To evaluate in vivo antioxidant and hepatoprotective activities of the methanolic extract of the root of Cassia singueana in rats following acute and chronic carbon tetrachloride intoxication. METHODS: Malondialdehyde (MDA), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and bilirubin as indices of liver damage and lipid peroxidation were detected in rats after intraperitoneal administration of extract (5 mg/kg). RESULTS: The liver, kidney and heart showed significant reduction (P<0.05) in the levels of MDA from (0.18±0.04), (0.23±0.07) and (0.26±0.10) nmol/mg respectively in the CCl4 control to (0.15±0.03), (0.17±0.04) and (0.17±0.07) nmol/mg protein in groups pre-treated with the extract for three days at 5 mg/kg). Similarly, compared to the CCl4 control, significant reduction (P<0.05) in serum AST, ALT and bilirubin as well as in level of total cholesterol and MDA with concomitant increase in HDL cholesterol, superoxide dismutase and catalase levels when CCl4-intoxicated rats were treated with Cassia singueana root extract for two weeks. CONCLUSIONS: These results suggest that methanolic extract of Cassia singueana contain potent antioxidant compounds that can offer significant protection against hepatic and oxidative injuries.

Hepatotoxic effects of chloroform extract of Artemisia macivera Linn in rats following intraperitoneal administration of different subchronic doses.

The possible hepatotoxic effects of chloroform extract of Artemisia maciverae was evaluated biochemically and histologically using male Swiss albino rats, randomly assigned into four groups of 24 animals each. The groups (control, 50, 100 and 200 mg/kg body weight) were treated for 60 days and then monitored for another 30 days before sacrifice. Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, bilirubin (total and direct), total protein and albumin were assessed colorimetrically, while tissue specimens were subjected to histological examination following standard hematoxyline-eosin staining techniques. After 1 week of treatment, the extract caused statistically significant elevation in levels of serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and bilirubin (total and direct), while there was significant (p < 0.05) decrease in the levels of serum total protein and albumin at the onset of treatment when compared with the control. These abnormalities in the levels of serum biochemical parameters were spontaneously corrected within 2 weeks of treatment. Similarly, histological assessment showed severe hepatic tissue injuries after 1 week, but these organs recovered spontaneously by the second week of treatment. The results indicate that long-term exposure to therapeutic doses of chloroform extract of A maciverae is relatively safe, but high dose exposure may result in hepatocellular injury.

Economic and Health Perspectives of Mycotoxins: A Review

Due to their potent toxicity; mycotoxins have attracted worldwide attention over the years and recently; there has been an increasing disquietness on the part of governments; producers; processors; marketers and consumers over the health and economic significance. The diversity in occurrence; structure and chemistry of mycotoxins make their impact more complex to diagnose. Owing to their usual environmental conditions of production in countries with warm and humid climates as well as poor conditions of storage and handling; agricultural commodities are susceptible to fungal colonization and development which can lead to the accumulation of mycotoxins. As part of drying process; agricultural produce are exposed to contamination by ubiquitous mycoflora that grow; develop and produce some toxic metabolites that are harmful to the consumers. Food is already a limited commodity; especially in developing countries of the world and consumers therefore; either as a matter of choice or for the relative cheapness and affordability during periods of scarcity; opt for the over-fresh produce; sometimes not aware of the adverse health implications such foods pose. In the quest to ensure regular and continuous availability of certain perishable farm produce; especially in developing nations; local farmers and traders resort to unscientific and faulty storage conditions to preserve commodities; thereby pre-disposing produce to fungal colonization and mycotoxin production. Thus; commodities such as groundnuts; maize; sorghum; rice; yam; cassava; tiger nut; soyabeans; cotton seeds; fruits; vegetables spices can be contaminated with toxins of fungal origin such as aflatoxins; ochratoxins; fumonisins; patulin; sterigmatocystin; deoxynivalenol; zearalenone and other mycotoxins which pose serious economic and health risks. This review presents some mycotoxins commonly found on agricultural commodities both in temperate and tropic regions of the world. The acute and chronic toxic effects of these toxins in humans and animals are highlighted. Control measures include education of the populace on the risks of exposure to mycotoxins through skin contact; inhalation and ingestion; early harvesting; rapid appropriate drying; sequestration of diseased seeds from sound seeds; sanitation; use of good agronomic practices; insect control; the use of botanicals and synthetics as storage protectants; biological control and detoxification of mycotoxin-contaminated commodities. Probable related health implications are also discussed with a view to creating better public awareness and providing scientific basis for appreciating the challenges; while proactively promoting the development and implementation of policies at mitigating risk factors. Some mycotoxins; their producer fungi and toxic effects are further presented

Changes in weight gain, faecal oocyst count and packed cell volume of Eimeria tenella-infected broilers treated with a wild mushroom (Ganoderma lucidum) aqueous extract.

An experiment was conducted to study the effect of a wild Ganoderma lucidum aqueous extract in coccidian-infected broilers. At 6 weeks of age the birds were randomly allocated to 6 treatment groups of 20 Ross broilers each in wire cages. Groups A, B and C were infected with Eimeria tenella Houghton strain at the rate of 36 250 sporulated oocysts/ml per bird. The remaining 3 groups D, E and F were uninfected controls. At 7 weeks the birds in group A were treated with G. lucidum aqueous extract and those in B with amprolium in drinking water ad libitum at the rate of 200 mg/ml each for 7 days consecutively. Body weight gain, feed intake, faecal oocyst output and some haematological parameters were monitored. The result showed that all the infected birds in groups A, B and C had clinical signs of weakness and reduced appetite on day 4 post-infection. By the 5th day post-infection their faeces became bloody and watery, and large numbers of E. tenella oocysts were present in the faeces. On day 3 after treatment the oocysts detected were considerably reduced in both treated groups A and B and slightly higher in the untreated group C. The faeces of the uninfected control groups were normal and free of coccidial oocysts. After treatment for 7 days no coccidial oocysts were found in faeces of the birds that had been treated. Infected, untreated birds showed a slight drop in feed intake and weight gain from 7 to 8 weeks of age. The final mean weight gain recorded in the treated groups A and B was comparable to that of the uninfected birds in the 3 control groups, while it was lower in the untreated group C. The feed to gain ratio was higher in C than in the other groups. A slight drop in packed cell volume was observed in groups A, B and C at 7 weeks of age, 1 week after infection. This study showed that treatment with G. lucidum results in a marked reduction in the number of E. tenella oocysts shed in the faeces, leading to improved weight gain and decreased weight loss. The results confirmed the virulence of the Houghton strain of E. tenella and the effectiveness of both amprolium and G. lucidum extract against E. tenella.

Bioassay-guided fractionation and in vivo antiplasmodial effect of fractions of chloroform extract of Artemisia maciverae Linn.

In the search for new plant-derived anti-malarial compounds, chromatographic fractions of chloroform extract of whole plants of Artemisia maciverae were tested in vivo using chloroquine resistant and chloroquine sensitive Plasmodium berghei NK 65 infected Swiss albino mice. One fraction and a sub-fraction of this were most active at 10/mg and cleared parasitemia in mice within 3 days. The different fractions and sub-fractions were tested with different reagents to determine the broad classes of compounds present. The active fraction tested positive for triterpenes and alkaloids, and the sub-fraction for only triterpenes. These tests suggest that the anti-malarial activities observed with these fractions may be due to these classes of compounds in the chloroform extract of the A. maciverae. Further chemical work is however required to characterize the active constituents.

Polyphenol composition and antioxidant potential of Hibiscus esculentus L. fruit cultivated in Nigeria.

Consumption of certain fruits and vegetables is now widely associated with chemoprevention of degenerative diseases like cancer and cardiovacsular disorders because of their antioxidant components. Polyphenols, a heterogeneous group of compounds, are one of these constituents. Hibiscus esculentus L. (Family Malvaceae), commonly referred to as okro, okra, or lady's finger, is an important component of diet in Nigeria and other countries in sub-Saharan Africa. In this article, we describe the polyphenol composition and antioxidant potential of H. esculentus of Nigerian origin. Quercetin glucoside (quercetrin) and an unidentified flavonoid were detected. In vitro antioxidant assay of methanol extract of the fruits showed potent antioxidant/radical scavenging activities with 50% inhibitory concentration values of 25 and 43 microL when analyzed by the xanthine oxidase and 2-deoxyguanosine methods, respectively. These data suggest that H. esculentus, popular especially during the rainy season in Nigeria and many tropical West, Central, and Eastern African countries, is a good contributor to the antioxidant status and disease chemoprevention of people in these countries.

Evaluation of the polyphenol composition and antioxidant activity of African variety of Dacryodes edulis (G.Don) H.J Lam fruit.

Polyphenols are abundant micronutrients in our diet that have been credited with chemoprevention of diseases associated with oxidative stress. In this study, we investigated the whole ripened fruit of Dacryodes edulis (G.Don) H.J Lam, a multipurpose tree growing in West and Central Africa and other countries bordering the Gulf of Guinea, for polyphenol content as well as its antioxidant/radical scavenging capacity. Analysis of the methanol extract of the fruit by high-performance liquid chromatography coupled to an ultraviolet dual-array detector and mass-selective detector revealed the presence of catechol (9.27 mg/kg), gallate (10.40 mg/kg), methylgallate (0.88 mg/kg), ellagic acid (3.10 mg/kg), quercetin (0.21 mg/kg), and quercetin rhamnoside (0.76 mg/kg). The extract showed very high antioxidant potential (50% inhibitory concentration [IC(50)] = 14 microL), but a rather weak radical scavenging activity (IC(50) = 357 microL), when tested in vitro with the xanthine oxidase and 2-deoxyguanosine assay model systems, respectively. These results suggest that consumption of D. edulis could contribute to prevention of diseases that are related to oxidative stress.

Antitrypanosomal effects of petroleum ether, chloroform and methanol extracts of Artemisia maciverae Linn.

Petroleum ether, chloroform and methanol extracts of A. maciverae were studied in vitro and in vivo for activity against Trypanosoma brucei brucei in Swiss albino mice. Thereafter, the chloroform extract which showed the highest activity in both in vitro and in vivo assessments was subjected to bioassay-guided fractionation. The crude extracts and the fractions of the chloroform extract of A. maciverae were screened for phytochemicals and secondary metabolites. Combined fractions 54-57 of this extract showed the highest in vitro antitrypanosomal activity, and at 10 mg/kg body weight, this fraction cleared the parasitemia completely from T. brucei brucei infected Swiss albino mice after 7 days of treatment. There was no statistically significant difference in the level of parasitemia when the infected mice treated with this fraction was compared with the standard trypanocidal drug, diminal. The results of the phytochemical analysis showed that the crude extracts contained secondary metabolites like flavonoids, triterpenes, terpenoids, tannins, phlobatannins and alkaloids, while the active fraction contains only triterpenes and alkaloids. It can be inferred that fraction 54-57 contains the active component responsible for the high antitrypanosomal activity of the chloroform extract of A. maciverae.

Assessment of the phytochemical and antitrypanosomal properties of some extracts of leaves, stem and root bark of Landolphia sp., P. Beauv.

UNLABELLED: INTRODUCTION/JUSTIFICATION: There is urgent need to source for alternative chemotherapy against trypanosmosis, a disease of major importance in human and economic animals. MATERIALS AND METHODS: Therefore, petroleum ether, chloroform, methanol and aqueous extracts sequentially obtained from the leaves, stem and root barks of Landolphia uniflora were evaluated for their in vitro and in vivo antitrypanosomal activities against Trypanosoma brucei brucei, as well as their phytochemical constituents. RESULTS: Steroids and triterpenes, resins, tannins, saponins and flavonosides were detected in almost all the extracts, but alkaloid was absent in methanol extract of the stem and stem, as well as the chloroform extract of the root bark. In vitro, all extracts of the roots displayed significant antitrypanosomal activity, while only the chloroform extracts of the leaves and stem bark showed activity at both test concentrations (4 and 2 mg/ml). However, under in vivo condition, the methanol extracts showed the greatest activity, eliminating parasitaemia within the 10 days treatment period and prolonging survival period at 200 and 300 mg/kg body weight intramuscular doses. CONCLUSION: These results suggest that Landolphia uniflora could be useful in the management of trypanosomiasis.

Occurrence of preformed volatile nitrosamines in preparations of some Nigerian medicinal plants: a preliminary report.

Preparations of some tropical plants of medicinal importance collected from the savannah vegetational belt of Nigeria were analysed for preformed volatile N-nitrosamines. N-nitrosamines were analyzed by chemiluminescence detection on a thermal energy analyzer (TEA) following gas chromatographic (GC) separation. Only N-nitrosodimethylamine (NDMA) in the range of 1.2-3.4 microg/kg was detected in four out of the 29 sample preparations. These preliminary data suggest that medicinal plant preparations may be due in part to microbial contamination, contributing to N-nitroso compound burden in many developing countries where ethnomedicine in still widely practised.

Indigenous knowledge system for treatment of trypanosomiasis in Kaduna state of Nigeria.

A survey was carried out in Kaduna State of Nigeria to establish the indigenous knowledge system for treating trypanosomiasis in domestic animals. Questionnaire and interviews were, respectively, administered to, or conducted with about 200 livestock farmers and traders spread around the state. Data obtained revealed the use of several plants either alone or in combination, for the treatment and management of trypasonomiasis. The most common plants encountered were Adansonia digitata, Terminalia avicennoides, Khaya senegalensis, Cissus populnea, Tamarindus indica, Lawsonia inermis, Boswellia dalzielli, Pseudocedrela kotschi, Syzyium quinensis, Sterculia setigera, Afzelia africana, Prosopis africana, Lancea kerstingii. The method of preparation and mode of administration of some of these plants in the treatment of trypanosomiasis are reviewed and discussed.

Cotinine effects on bile flow and biliary NNK elimination.

Nicotine and its major metabolite cotinine inhibit alpha-hydroxylation of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) suggesting that an alternative pathway of NNK metabolism and elimination, biliary excretion of the O-glucuronide of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL-Gluc) may be enhanced. To verify the possible role of cotinine on biliary elimination of NNK and its metabolites, bile duct cannulated rats were administered a single i.p. dose of 50 mg/kg [56sup;-3 H]-NNK with or without i.p. co-administration of 5 mg/kg cotinine or nicotine. Cotinine significantly reduced cumulative bile flow and biliary elimination of NNK-derived radioactivity within six hours to 42 and 27 percent, respectively. The pattern of NNK metabolites in bile was unchanged. Nicotine had a similar inhibitory effect on bile flow. This result constitutes the first experimental evidence that cotinine inhibits bile flow. In rats, biliary elimination of NNK is reduced accordingly which may lead to an increased carcinogen burden in the body. In humans, inhibition of bile flow by tobacco alkaloids may contribute to the appetite suppressing effect of tobacco products.

4-Hydroxy-1-(3-pyridyl)-1-butanone-hemoglobin adducts as biomarkers of exposure to tobacco smoke: validation of a method to be used in multicenter studies.

Hemoglobin (Hb) adducts of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB), a metabolite of two tobacco-specific nitrosamines [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine], were measured as biomarkers of exposure to tobacco smoke as part of a study on genetic alterations and susceptibility to lung cancer among nonsmokers. HPB-Hb adducts were measured after collection of RBCs by Ficoll gradient in six collaborating centers, release of HPB by alkaline hydrolysis from Hb, clean-up by solid-phase extraction, and analysis of an electron-capturing derivative by gas chromatography-electron capture mass spectrometry. Prior to analysis of samples from study subjects, the reproducibility of this approach was validated in blood from donors. The coefficient of variation of reproducibility of paired aliquots from five samples ranged from 7 to 25%; the within-sample reproducibilities of four and eight aliquots were 4 and 16%, respectively. The study subjects consisted of 18 smokers and 52 never-smokers. HPB-Hb adduct levels were significantly higher (P = 0.02) in smokers (26 +/- 13 fmol HPB/g Hb) than in never-smokers (20 +/- 8 fmol HPB/g Hb). There was no difference between sexes. These results suggest that the level of HPB-Hb adducts, measured using a method modified to facilitate use in multicenter studies, can be a useful biomarker of exposure to tobacco smoke.

Bacterial reduction of N-oxides of tobacco-specific nitrosamines (TSNA).

1. Contrary to established metabolic pattern, a recent investigation of NNK metabolism produced in rat urine higher levels of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) than their N-oxides, suggesting that reconversion of N-oxides could occur after urine formation. 2. To verify the possible role of bacteria in the reduction of NNK-N-oxide and NNAL-N-oxide to their respective parent compounds, NNK and NNAL, in smokers with urinary tract infection (UTI), the N-oxides were isolated from the urine of rats treated with [5-3H]NNK and individually incubated at 37 degrees C with ten bacterial species in sterile human urine under different pH regimens. After incubation with the bacteria, aliquots of culture media were analyzed by high pressure liquid chromatography (HPLC) with radiochemical detection. 3. Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae and Proteus mirabilis possessed varying capacity to regenerate NNK and NNAL from their N-oxides while others showed no detectable reductive capability within 24 h. 4. This result constitutes the first experimental evidence that in tobacco users with concomitant UTI, bacterial regeneration of the procarcinogenic NNK and NNAL from their N-oxides could occur in the bladder leading to increased carcinogen burden in these individuals.

Detection of methylating activity due to nitrosamide in some nitrosated Nigerian foodstuffs.

Popular vegetables, condiments and some Nigerian staple foods were evaluated for their relative methylating potential due to nitrosamide formation following nitrosation under standardized conditions. Methylating activity of nitrosated foodstuffs, expressed as N-nitroso-N-methylurea equivalents, was determined by gas chromatography-thermal energy analysis. In positive samples (detection limit 10 microgram/kg) methylating activity detected was in the range of 50-1200 microgram/kg, the highest activity being found in Telfairia occidentalis (ugwu). This value constitutes the highest amount ever detected for a fresh vegetable. The data suggest that some commonly consumed local foodstuffs might contribute to overall human burden of environmental carcinogens in Nigeria.

Tobacco-specific nitrosamines in some Nigerian cigarettes.

Ten popular brands of cigarettes on the Nigerian market were analyzed for tobacco-specific nitrosamines (TSNA) in tobacco and in mainstream smoke, as well as nitrate in tobacco. TSNA was analyzed using a gas chromatography/thermal energy analyzer (GC-TEA), while nitrate was determined spectrophotometrically as nitrite following on-line reduction with copper, diozatization with sulfanilamide and coupling with N-(1-naphthyl) ethylene diamine to form an azo dye. In mainstream smoke, the concentration of NNN, NAB/NAT and NNK were respectively, between 8 and 90 ng, 10 and 65 ng, and between 15 and 72 ng/cigarette. Preformed NNN ranged between 64 and 565 ng/cigarette, while preformed NAB/NAT and NNK ranged respectively from 109 to 476 ng/cigarette, and from 55 to 317 ng/cigarette. Nitrate levels ranged between 1.5 and 6.1 mg/g tobacco. In general, the results indicate that the TSNA content of Nigerian cigarettes are within the range found for European and American cigarettes.

Nitrosatable amines and nitrosamide formation in natural stimulants: Cola acuminata, C. nitida and Garcinia cola.

Three varieties of kola nut, Cola acuminata, C. nitida and Garcinia cola, of Nigerian origin, were analysed for their content of primary and secondary amines, and assessed for their relative methylating potential due to nitrosamide formation. Primary and secondary amines were determined as benzene sulfonamides by gas chromatography/thermal energy analysis (GC/TEA). Dimethylamine, methylamine, ethylamine and isopentylamine were detected in all kola nut varieties, while pyrrolidine, piperidine and isobutylamine were detected in one or more varieties. Estimated average total daily intake of aliphatic amines by a typical kola nut chewer varied from 260 to 1040 micrograms/day for secondary amines and from 2430 to 9710 micrograms/day for primary amines. Methylating activity of the nitrosated kola nuts, expressed as N-nitroso-N-methylurea equivalents, was also determined by GC/TEA. Methylating activity was significantly higher in kola nuts (170-490 micrograms/kg) than has ever been reported for a fresh plant product. These data suggest that the possible role of kola nut chewing in human cancer aetiology should be explored in countries where kola nuts are widely consumed as stimulants.

Nitrosation of Nigerian medicinal plant preparations under 'chemical' and 'simulated' gastric conditions.

Preparations of some tropical plants of medicinal importance, collected from the savannah vegetational belt of Nigeria, were nitrosated and analysed for volatile N-nitrosamines formed under chemical and simulated gastric conditions. N-Nitrosamines were determined on a Thermal Energy Analyser following gas chromatographic separation. Mean concentrations of N-nitrosodimethylamine (NDMA) in the range of 7 to 58 ppb and N-nitrosodiethylamine (NDEA) in the range of 23 to 26 ppb were formed in 31 and 7%, respectively, of the preparations using artificial gastric juice (simulated gastric condition). Under chemically optimal conditions, relatively high levels of NDMA (72-2008 ppb), NDEA (23-1528 ppb) and N-nitrosopyrrolidine (20-405 ppb) were formed in 100, 75 and 32% of the preparations, respectively; N-nitrosomethylethylamine, N-nitrosodibutylamine and N-nitrosomorpholine were formed in fewer preparations. These findings suggest that the endogenous formation of N-nitroso compounds from precursors present in medicinal plants might be another source of human exposure to environmental carcinogens in Nigeria and other developing countries.

Precursors of N-nitroso compounds in some Nigerian medicinal plants.

Twenty-seven tropical plants of medicinal importance were analysed for primary and secondary amines by chemiluminescence detection on a Thermal Energy Analyzer (TEA) modified for use on 'nitrogen mode' following derivatization with benzene sulphonyl chloride (BSC) and gas chromatographic (GC) separation of their sulphonamides. Nitrite was determined by colorimetry at 540 nm after diazotization with sulphanilamide and coupling with N-(1-naphthyl)ethylenediamine to form an azo dye. Nitrate was determined as nitrite following on-line reduction by granulated cadmium. Dimethylamine in the range of 0.5 ppm to 18.2 ppm was detected in 96% of samples, while pyrrolidine ranged between 0.7 ppm and 12.78 ppm in 14 samples. Isobutylamine, methylamine and ethylamine were the most ubiquitous primary amines. Largest number of secondary amines (four) was found in Azadirachta indica (Neem) while largest number of primary amines (six) was detected in Azadirachta indica and Tamarindus indica (Tsamiya) which also contained the highest amount of total primary amines (148.8 ppm). Nitrate and nitrite were seldom found in plant extracts whose pH were generally below 7.0. These findings suggests that early exposures to precursors of N-nitroso compounds via medicinal plants might contribute to total risk posed by environmental carcinogens in Nigeria.

Metabolism of a glucuronide conjugate of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in rats.

Besides 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), [4-(methylnitrosamino)-1-(3-pyridyl)butl-yl]-beta-O-d-glucosidu ronic acid (NNAL-Glu) is another important metabolite of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) which has been detected in the urine of tobacco users and non-smokers heavily exposed to sidestream cigarette smoke. In order to evaluate the toxicological significance of NNAL-Glu formation and excretion, the metabolism of [5-3H]-NNAL-Glu was studied in rats. Five male F344 rats were administered 3.7 mg/kg [5-3H]-NNAL-Glu by i.v. injection and the metabolites in urine analysed by HPLC. More than 90% of the radioactivity was excreted in urine within the first 24 h. Unchanged NNAL-Glu accounted for 81.2 +/- 3.1% of the total radioactivity; the remaining part of the dose appears to be deconjugated resulting in the urinary excretion of NNAL (3.6 +/- 1.7%) and its alpha-hydroxylation (11.5 +/- 2.2%) and N-oxidation (3.6 +/- 1.6%) products. The presence of alpha-hydroxylation products of NNAL-Glu in urine suggests that this NNK metabolite may be activated in vivo to carcinogenic intermediates.