Dietary vitamin D ameliorates hepatic oxidative stress and inflammatory effects of diethylnitrosamine in rats.

The generation of reactive oxygen species (ROS) plays an essential role in the pathogenesis of several diseases. Its implication in inflammation has suggested a possible link between oxidative stress and activation/release of cytokines in precancerous states. Recent observational studies have suggested an association between inflammation and vitamin D deficiency; hence, suggesting that vitamin D could play a role in the pathogenesis of diseases. This study examined the antioxidant and anti-inflammatory potentials of vitamin D in diethylnitrosamine (DEN)-induced oxidative stress and inflammation in rats. Rats were divided into four experimental groups. While groups one and two were administered twice weekly with 30 mg/kg body weight DEN for six weeks, groups three and four were given normal saline. Groups one and three were fed with vitamin D deficient diet, while groups two and four were fed vitamin D diet during the experiment. After that, biomarkers of oxidative stress status were assayed spectrophotometrically. The concentration of inflammatory cytokines was determined using enzyme-linked immunosorbent assay (ELISA). DEN-induced vitamin D deficient diet group had increased antioxidant enzymes' activities. Also, there were elevated concentrations of thiobarbituric acid reactive substances (TBARS) and inflammatory cytokines in the same group. Vitamin D diet, however, reduced oxidative stress effects through the reduction in the activities of TBARS and caused a significant (p < 0.05) increase in nitric oxide concentration. Vitamin D diet significantly (p < 0.05) reduced the level of interleukin 1ß and TNF-α produced in the deficiency state. These findings show that vitamin D may play an essential role in the regulation of hepatic oxidative stress and inflammatory responses.

Toxicokinetics and Biliary Excretion of N-Nitrosodiethylamine in Rat Supplemented with Low and High Dietary Proteins.

Although biliary excretion is one of the biological elimination processes for foreign compounds, intake of high-protein diets was hypothesized to enhance their detoxification rates. Hence, this study investigates the effect of differential dietary protein intake on toxicokinetics and biliary excretion in rats following exposure to N-nitrosodiethylamine (NDEA) and aflatoxin B1 (AFB1). The animals were divided into five groups. Groups I and II were exposed to low and high dietary proteins following a single intraperitoneal dose of 43 µg NDEA/kg body weight, respectively. Groups III and IV were equally treated after a combined single intraperitoneal dose of 43 µg NDEA plus 0.022µg AFBI/kg body weight, respectively. Group V was fed with low-protein diets following a single intraperitoneal dose of 0.022µg AFB1/kg body weight. The experiment lasted 35 days. The bile excreted higher amounts of unchanged NDEA than nitrite. The groups placed on high-protein diets (HPD = 64%) eliminated higher amounts of the unchanged NDEA and nitrite than the lower-protein diet (LPD = 8%) groups. Furthermore, the animals fed with high dietary protein (HPD = 64%) depicted short half-life with corresponding increase in elimination rate constant. The presence of AFB1 heightened the excretion of bound NDEA with AFB1 than NDEA only. Generally, this study advocates that N-nitrosodiethylamine and the corresponding metabolites follow hepatobiliary system potentiated by high intake of dietary proteins.

Antioxidant Effects of Methanol Extract of Allium cepa linn on Cyanide-induced Renal Toxicity in Male Wistar Rats.

The protective effects of onion was assessed in the Kidney of rats following sub-acute exposure to cyanide. These effects were compared to those of sodium thiosulphate (Na2S2O3), a classical antidote of cyanide toxicity. The rats were divided into 6 groups of 6 animals each. Group 1 was administered distilled water, Group 2,3,4, 5, and 6 were administered 600 mg onion/kg bwt/day, 7 mg KCN/kg bwt/day, 300 mg onion/kg bwt/day+ 7 mg KCN/kg bwt/day, 600 mg onion/kg bwt/day+ 7mgKCN/kg bwt/day, 600mg Na2S2O3/kg bwt/day+ 7mgKCN/kg bwt/day respectively for 2 weeks. Group 3, 4 and 5 were pre-administered with 300mg onion/kg bwt/day, 600mg onion/kg bwt/day and 600mg Na2S2O3 /kg bwt/day respectively for 2 weeks. Serum and urine creatinine and urea level were assessed as a measure of kidney function. Oxidative stress and antioxidant parameters were estimated in Kidney. Serum creatinine and urea levels were significantly higher in the cyanide treated rats compared with control. This was accompanied by significant reduction in the urine level of creatinine and urea. Co-administration with onion extract and Na2S2O3 reverse the situation in both the serum and urine. The level of malondialdehyde (MDA) in rats treated with cyanide (3.846±0.20µg/g) was significantly increased in the kidney relative to control (0.691±0.15µg/g). This was accompanied with a decreased in antioxidant enzymes Superoxide Dismutase (SOD) (2.0±0.09U/mg), Catalase (CAT) (0.014±0.001katf),Glutathione-S-Tranferase (GST) (0.015±0.009nMol/mg) and non-enzymatic antioxidant Reduced Glutathione (GSH) (4.006±0.09µg/ml) compared with control (4.8±0.13 U/mg, 0.047±0.001 katf, 0.022±0.0013 nMol/mg, 6.802±0.2 µg/ml respectively). Co-administration with onion extract and Na2S2O3 significantly increased these antioxidant enzymes and significantly decreased the concentration of malondialdehyde in the kidney. The results indicate that onion extract reduced lipid peroxidation in the kidney and increased antioxidant status of animals exposed to cyanide in a dose dependent manner.

Pharmacokinetics of biliary excretion of N-nitrosodimethylamine in rats fed diets containing levels of protein.

Albino Wistar rats (Rattus norvegius) fed semi-purified diets containing 3.5%, 8%, 27%, and 64% casein, respectively, as the protein source, were poisoned with an intraperitoneal dose of 20mg N-nitrosodimethylamine (NDMA)/kg, following cannulation of the bile duct, in vitro, under urethane anaesthesia. Bile exudates was collected at designated time intervals and analysed for unchanged NDMA using thin layer chromatography and gas liquid chromatography methods. Rats on 64% high protein diet (HPD) were the highest excretors of NDMA, followed by rats on the 3.5% kwashiorkorigenic diet (KWD), 8% low protein diet (LPD) and 27% normal protein diet (NDP) as the least excretors, in that order. The corresponding values for culmulative excretions of NDMA were 4.38%, 2.74%, 2.96% and 4.11%, and for elimination rate contents they were 54.05Kh(-1), 23.01Kh(-1), 23.76Kh(-1) and 48.88Kh(-1), while the respective elimination half-life values were 0.013h, 0.031h, 0.029h and 0.014h. The toxicological and pharmacological implication of the pharmacokinetic findings are discussed.

Occupational and dietary exposures of humans to cyanide poisoning from large-scale cassava processing and ingestion of cassava foods.

The biochemical and toxicological effects of occupational and dietary exposure of humans to cyanide poisoning from large-scale cassava processing and ingestion of cassava foods were investigated using spectrophotometric and enzymatic methods. Analysis of urinary and serum thiocyanate (cyanide metabolite) from workers in cassava processing industries, who were 'frequent' [those who eat cassava food(s) at least once a day] and 'infrequent' [those who eat cassava food(s) only occasionally] consumers of cassava-based diets, was carried out with the aid of questionnaries. The mean urinary thiocyanate level of the cassava processors (mean+/-S.D.; 153.50+/-25.21 micromo1/l) was 2.2 and 2.6 times higher than that of frequent (70.1+/-21.8 micromo1/l) and infrequent (mean+/-S.D.; 59.30+/-17.0 micromo1/l) cassava consumers, respectively. The mean serum thiocyanate levels rose to 126.73+/-12.4 micromo1/l for the former and 68.4+/-18.3 and 54.7+/-13.2 micromo1/l, respectively, for the latter. An increase in plasma activity by 10% above normal of aspartate aminotransferase (AST) was observed in 40% of the cassava processors, whereas it was within normal range in all consumers. The activities of alanine aminotransferase (ALT) and alkaline phosphatase (ALK.PHOS) were within the normal value in all cases studied. The blood glucose level of 50% of the cassava processors was 100 mg/ml or above while that of the consumers was in the range of 68-85 mg/100 ml. The total protein, serum albumin and creatinine levels were in the range for normal values for the processors and consumers. The health implications of these findings are discussed.

Preformed volatile nitrosamines in some Nigerian foodstuffs.

Some common Nigerian foodstuffs were assessed for their content of preformed volatile nitrosamine by chemiluminescence detection following gas chromatographic separation. Nitrosodimethylamine levels of between 0.4 and 4.6 ppb were detected in 75% of the samples analysed. The highest value was found in Brassica oleraceae, while Vernonia amygdalina contained the lowest detectable level. These data suggest that Nigerians may be exposed to chronic but very low levels of carcinogenic nitrosamines in their foods.

Potential of endogenous formation of volatile nitrosamines from Nigerian vegetables and spices.

Nitrosatability of dried Nigerian vegetables and spices was investigated under simulated gastric conditions. N-Nitroso-dimethylamine (NDMA) was the only volatile nitrosamine found above the preformed level except in ugwu where N-nitroso-piperidine (2.3 ppb) was also detected. The lowest NDMA value of 0.4 ppb was found in bitterleaf while onions had the highest level (14.7 ppb) of nitrosation. The results suggest that, under endogenous conditions, nitrosation of these foodstuffs could be an important contributory factor in cancer aetiology.

Pharmacokinetics of biliary excretion of N-nitrosodiphenylamine (NDPA) in animals of different species.

Pharmacokinetic investigations into the biliary excretion of N-nitrosodiphenylamine given as an i.p. dose of 50 mg/kg were conducted and results compared in three animal species; rat, guinea pig and rabbit following bile duct cannulation and collection of bile in vitro. The guinea pig excreted NDPA into bile fastest while the rabbit, which excreted it slowest, eliminated it fastest. Both appearance and disappearance of the nitrosamine were comparatively slow in the rat. NDPA elimination half-life values in the animal species were 510, 240 and 95 min respectively, while cumulative excretions amounted to 12, 3 and 0.3 percent. The toxicological implications of these species differences are highlighted.

Metabolism of linamarin in rats.

The metabolism of linamarin [2(beta-D-glucopyranosyloxy)isobutyronitrile] was investigated in male albino Wistar rats and using rat liver microsomal preparations. In the in vitro experiments incubations of varying concentrations of linamarin at pH 6.0-6.5 with liver microsomal preparations resulted in rapid degradation of the substrate without concomitant production of any detectable amount of hydrogen cyanide (HCN) or of thiocyanate, its detoxication derivative. Boiled incubation medium did not degrade linamarin. Mathematical treatment of the degradation data generated theoretical HCN values that were used to construct a Lineweaver-Burke plot, which gave apparent Km and Vmax values of 3.3 mM-linamarin and 0.017 mg HCN/min/mg protein, respectively. In the in vivo experiments excretion of glucosidic cyanide (linamarin) in rat urine was found, within the range of applied oral doses 10-350 mg/kg body weight, to be dose dependent. Urinary excretion of HCN and thiocyanate did not show this correlation. Following administration (iv) of 10, 50 or 100 mg linamarin, elimination of the test substance from rat blood was observed to occur exponentially, and the half-life was estimated at about 90 min for all three dose levels.

Nitrosamine metabolism in protein-deficient weanling rats during the process of repletion.

The metabolism of N-nitrosodimethylamine (NDMA) during a 5- to 10-day process of 20% protein repletion was investigated, in vitro and in vivo, in male weanling albino Wistar rats acutely and chronically deprived of protein by feeding, respectively, diets containing no protein or 3.5% for 7 days and one with 3.5% protein for 35 days. Results of experiments using the 5-day rehabilitated animals showed that rate of NDMA demethylation by liver microsomal preparation was 3-fold higher in the chronically protein-dependent animals but comparable between control animals and the more acutely deprived species. In addition, urinary excretion of an oral dose of 40 mg NDMA/kg was significantly less in the chronically protein-dependent animals, which also cleared the blood of an intravenous dose of 5 mg NDMA/kg more rapidly. NDMA metabolism was comparable in all animal groups after 10 days of protein replection.

Dietary thiocyanate and N-nitrosation in vivo in the Wistar rat.

The influence of dietary thiocyanate (SCN-) on N-nitrosation in vivo was investigated over 14 min following the administration, by stomach tube, of single doses of sodium nitrite and dimethylamine hydrochloride to male albino Wistar rats whose diet contained appreciable quantities of bound cyanide (cyanogenic glycosides) and free (nonglycosidic) cyanide. The rate of disappearance of the nitrosating agent (NO2-) from the stomach in control animals showed a high linear correlation (r = -0.84) with gastric N-nitrosation, as measured by metabolism (N-demethylation) of 'formed' nitrosamine in liver tissue isolated from these animals. This statistical relationship was significantly increased (r = 0.98), as well as the rate of NO2- utilization and the activity of the N-demethylase enzyme, in animals fed the test diet. Stomach SCN- content was well correlated with both stomach NO2- concentration (r = -0.95) and liver N-demethylase activity (r = +0.93). Interactions, in vivo, between thiocyanate ion and nitrosamine precursors ingested in food may enhance nitrosamine carcinogenesis.

Biliary excretion of linamarin in the Wistar rat after a single dose.

The biliary excretion of linamarin (2[beta-D-glucopyranosyloxy]isobutyronitrile) was studied in male albino Wistar rats injected i.p. with single doses of 300 mg linamarin/kg following cannulation of the bile duct in vivo; 24 hr faeces of uncannulated rats, similarly dosed, was examined for excretory products. Enzymatic and spectrophotometric analyses of the bile exudate showed that glucosidic cyanide (linamarin, and non-glucosidic cyanide were excreted; the elimination of both cyanide forms exhibits biphasic kinetics. Thiocyanate ion was undetectable. T.l.c. of the test bile followed by enzymatic and chemical investigation of the chromatogram confirmed the presence of unchanged linamarin, and four different u.v. fluorescent non-glucosidic cyanide metabolites. Neither linamarin nor cyanide ion was detectable in faeces of the uncannulated rats.

N-nitrosamines and Nigerian habitual drinks, and cancer.

Commercially available samples of Nigerian lager beers and bottled palm wine, and of root cuttings of medicinal plants, assayed for volatile N-nitroso compounds and nitrosatable entities, respectively, by chromatographic, chemiluminescence, colorimetric, derivatisation and nitrosation methods, contained nitrosodimethylamine (0.2-2.3 micrograms/kg; mean, 0.9 micrograms/kg) only in the drinks, and dimethylamine (DMA) and piperidine (1 mg/kg as DMA) in the roots.

Nitrosation of quaternary ammonium compounds in vivo in the Wistar rat.

Animals dosed orally with nitrite (1.5 X 10(-3) mol/kg) and nonyl dimethylbenzylammonium bromide (2.9 X 10(-4) mol/kg) exhibit liver damage within two hours; cetyl trimethylammonium bromide plus nitrite was not significantly hepatotoxic. Both nonyl dimethylbenzyl ammonium bromide and cetyl trimethylammonium bromide were demethylated by rat-liver microsomal preparations; substrate concentrations of 1 mM or more were inhibitory. Bile from rats given i.p. doses of cetyl trimethylammonium bromide, nonyl dimethylbenzylammonium bromide or dodecyl dimethylbenzylammonium bromide contains only metabolites, no unchanged compounds were detected.

Effect of thiamin status on the metabolism of linamarin in rats.

The effect of graded levels of thiamin on the metabolism of linamarin was investigated in rats. It was observed that on a diet deficient in thiamin, a large amount of linamarin was recovered unchanged in the urine, together with significantly more thiocyanate (SCN-) relative to the control. The least amount of thiocyanate (p less than 0.05 relative to control) was found in animals receiving the highest amount (twice daily requirement) of thiamin in the diet; but the amount of unmetabolized linamarin was similar to the control. It is suggested that thiamin deficiency may be implicated in the aetiology of tropical ataxic neuropathy (TAN) through the thiocyanate overload in people eating large amounts of cassava and cassava derivatives which contain linamarin.

Nitrosamine metabolism in kwashiorkor rats.

The in vitro metabolism of nitrosodimethylamine (NDMA) was studied in liver tissue obtained from male weanling kwashiorkor wistar rats. The elimination of this compound and that of nitrosomorpholine (NMOR) from the blood, after a single intravenous dose, was also investigated. N-demethylase activity in liver microsomes of the test animals was not significantly different from that of the controls although the activity of this enzyme per gram wet liver tissue was considerably reduced in the model animals. On the other hand, the glutathione (GSH) content in liver cytosol of the kwashiorkor animals was much higher than that of the controls. The elimination of NDMA and NMOR from the blood of the experimental animals over 8 hr following i.v. administration of the carcinogens, showed that the clearance rate of each nitrosamine was significantly lower in the kwashiorkor rats.

Differential effects on the cyanogenic glycoside content of fermenting cassava root pulp by beta-glucosidase and microbial activities.

The degradation of cyanogenic glycosides was studied in spontaneously fermenting cassava root pulp and in fresh pulp samples pretreated to prevent either endogenous beta-glycosidase activity, fermentation, or both. The rate of disappearance of the glycosides, as measured by hydrocyanic acid (HCN) production in situ, in membrane-sterilised media or in samples containing 1% sodium iodoacetate, was comparable with the untreated control in which 85% of the substrate was broken down within 72 h. Pretreatment of the fresh pulp with the beta-glucosidase inhibitor 1,5-gluconolactone (1%) markedly reduced the rate of disappearance of the cyanogens while inclusion of glucose in this test medium at the 3% level appeared to induce some hydrolysis. Loss of bound (glycosidic) cyanide in sterilised medium containing the glucosidase inhibitor was negligible. The results suggest that the contribution of the fermentation process in cyanide detoxification of pulped cassava roots is minimal.

Detoxification of cassava leaves by simple traditional methods.

Cassava leaves, which usually contain large quantities of cyanogenic glycosides, were processed into a Zairian traditional vegetable sauce "Mpondu" by simple methods which included blanching (10 min), mashing and then boiling for 20-80 min. These methods enhanced the detoxification of the leaves, with blanching alone resulting in the loss of 57% of the free (non-glycosidic) cyanide content and of 60% of the bound (glycosidic) cyanide. It is presumed that losses of cyanide during these processes would be accounted for in volatile HCN, its derivatives and in the boiling water.