Effects of green and black tea biocomposites on endogenous synthesis, metabolism and genotoxic effect of carcinogenic N-nitrosodimethylamine.

AIM: To study the modifying effect of green and black tea biocomposites on endogenous synthesis and genotoxic action of the carcinogenic N-nitrosodimethylamine. METHODS: Green and black tea biocomposites were administered to the white inbred rats in vivo. Amidopyrine and sodium nitrite were used as N-nitrosodimethylamine precursors and 4-methylpyrazol as an inhibitor of its metabolism. N-nitrosodimethylamine (blood, daily urine and reaction mixture), nitrites and nitrates (daily urine) levels were measured. Genotoxic action was tested by formation of DNA single-strand breaks in hepatocytes. RESULTS: In in vitro system, biocomposites increased N-nitrosodimethylamine synthesis in neutral medium and decreased in acid conditions. In vivo, black tea biocomposite consumption resulted in enhanced background level of DNA single-strand breaks in rats hepatocytes and higher genotoxic effect upon administration of N-nitrosodimethylamine precursors. The levels of N-nitrosodimethylamine in blood and urine of experimental animals were increased after precursors' administration. In contrast, green tea biocomposite significantly decreased background level of DNA single-strand breaks. However, there was no protective action of this food supplement at the N-nitrosodimethylamine, precursors' administration. 4-methylpyrazol administration did not increase N-nitrosodimethylamine excretion in urine, while this effect was observed in control and black tea biocomposite groups. CONCLUSIONS: The effects of green tea and black tea biocomposites on N-nitrosodimethylamine synthesis in in vitro system are unidirectional and depend on biocomposites' concentration and acidity of the medium. Long-term consumption of black tea biocomposite resulted in intensification of endogenous N-nitrosodimethylamine synthesis and increased damage of the hepatocytes' DNA. As to the green tea biocomposite, the obtained results allow us to suggest that this biocomposite enhanced N-nitrosodimethylamine metabolism.

Intragastric formation and modulation of N-nitrosodimethylamine in a dynamic in vitro gastrointestinal model under human physiological conditions.

Human exposure to carcinogenic N-alkylnitrosamines can occur exogenously via food consumption or endogenously by formation of these compounds through nitrosation of amine precursors. Information on the intragastric formation of NDMA from complex mixtures of precursors and inhibitors in humans is not available. In this study the formation of N-nitrosodimethylamine (NDMA) has been quantitatively analysed in a dynamic in vitro gastrointestinal model, in which gastric conditions can be modulated and closely simulates the physiological situation in humans. Substantial amounts of NDMA were produced when nitrite and dimethylamine or codfish were simultaneously introduced into the model. However, humans are gradually exposed to nitrite by the intake of nitrate-containing food. Nitrate secreted in saliva is converted to nitrite by oral bacteria. To mimic the human exposure to nitrite in a realistic way, nitrite was gradually added into the gastric compartment, simulating the swallowing of nitrite containing oral fluid after the intake of nitrate at the level of 0.1-10 times the ADI. Under these conditions, the cumulative amounts of NDMA formed were 2.3-422 microg NDMA and 1.8-42.7 microg NDMA at a rapid and slow gastric pH decrease, respectively. Beside codfish, various fish species and batches in combination with nitrite, simulating the intake of for times the ADI of nitrate, were investigated. Herring, pollack and plaice were also able to induce NDMA formation. Mackerel, salmon and pike perch did not result in increased NDMA formation. Furthermore, the effect of nitrosation modulators on NDMA formation was investigated. Thiocyanate (2 mM) increased NDMA formation, but the increase was not statistically significant. In contrast, orange jus and tea effectively, but not totally, reduced the amount of NDMA formed in the gastric compartment. These experiments show that (1). the dynamic in vitro gastrointestinal model is an appropriate tool for mechanistic studies on the intragastric formation of nitrosamines, and (2). that the results obtained with this model are helpful in evaluating human cancer risk for the combined intake of codfish-like fish species and nitrate-containing vegetables.

Inhibitory effect of whole strawberries, garlic juice or kale juice on endogenous formation of N-nitrosodimethylamine in humans.

In vitro and in vivo experiments were performed on inhibition of nitrosation by strawberry, garlic, and kale extracts. Strawberry, garlic, and kale extracts inhibited nitrosation in vitro. However, garlic extract has a greater ability to inhibit the chemical nitrosation in vitro than strawberry and kale extracts. The garlic methanol-soluble fraction of the garlic extract was fractionated into G1-G4 fractions by Prep-LC. Fraction G1 inhibited N-nitrosodimethylamine (NDMA) formation by 84+/-1%. We studied the formation of the carcinogen NDMA in humans after administration of nitrate (400 mg/day) in combination with an amine-rich diet and its possible inhibition by administration of whole strawberries (300 g), garlic juice (200 g: 75 g garlic juice in drinking water), or kale juice (200 g) in 27 males and 13 females (ten healthy volunteers in each group) of age 24+/-3 years. Nitrate intake resulted in a significant rise in mean salivary nitrate and nitrite concentrations. Also, nitrate excretion in urine during the experimental day was significantly increased compared with the control days. When whole strawberries, garlic juice, or kale juice was provided immediately after an amine-rich diet with a nitrate, NDMA excretion was decreased by 70, 71, and 44%, respectively, compared with NDMA excretion after ingestion of an amine-rich diet with a nitrate. These results suggest that consumption of whole strawberries, garlic juice, or kale juice can reduce endogenous NDMA formation.

Effect of ascorbic acid and green tea on endogenous formation of N-nitrosodimethylamine and N-nitrosopiperidine in humans.

Many constituents present in the human diet may inhibit endogenous formation of N-nitroso compounds (NOC). Studies with human volunteers showed inhibiting effects of intake of ascorbic acid and green tea consumption on nitrosation using the N-nitrosoproline test. The aim of the present study was to evaluate the effects of ascorbic acid and green tea on urinary excretion of carcinogenic N-nitrosodimethylamine (NDMA) and N-nitrosopiperidine (NPIP) in humans. Twenty-five healthy female volunteers consumed a fish meal rich in amines as nitrosatable precursors in combination with intake of nitrate-containing drinking water at the Acceptable Daily Intake level during 7 consecutive days. During 1 week before and after nitrate intake a diet low in nitrate was consumed. Using the same protocol, the effect of two different doses of ascorbic acid (250 mg and 1 g/day) and two different doses of green tea (2 g and 4 g/day) on formation of NDMA and NPIP was studied. Mean nitrate excretion in urine significantly increased from control (76+/-24) to 167+/-25 mg/24 h. Intake of nitrate and fish resulted in a significant increase in mean urinary excretion of NDMA compared with the control weeks: 871+/-430 and 640+/-277 ng/24 h during days 1-3 and 4-7, respectively, compared with 385+/-196 ng/24 h (p<0.0002). Excretion of NPIP in urine was not related to nitrate intake and composition of the diet. Intake of 250 mg and 1 g of ascorbic acid per day resulted in a significant decrease in urinary NDMA excretion during days 4-7 (p=0.0001), but not during days 1-3. Also, consumption of four cups of green tea per day (2 g) significantly decreased excretion of NDMA during days 4-7 (p=0.0035), but not during days 1-3. Surprisingly, consumption of eight cups of green tea per day (4 g) significantly increased NDMA excretion during days 4-7 (p=0.0001), again not during days 1-3. This increase is probably a result of catalytic effects of tea polyphenols on nitrosation, or of another, yet unknown, mechanism. These results suggest that intake of ascorbic acid and moderate consumption of green tea can reduce endogenous NDMA formation.

[Vitamin A and enzyme systems of metabolic activation of genotoxic compounds]. / Vitamin A i fermentnye sistemy metabolicheskoi aktivatsii genotoksichnykh soedinenii.

The study was undertaken to study the effects of N-nitrosodimethylamine (NDMA) on the formation of single-strand DNA breaks and gamma-glutamyltransferase-positive knots, the status of the enzymatic systems involved in NDMA metabolism and some other biochemical parameters when rats were on retinol-deficient diets and when they were given excessive vitamin A. The action of retinol on NDMA effects were analyzed by evaluating the activity of glutathione-S-transferase (EC 2.5.1.18), glutathione-reductase (EC 1.2.1.1), aldehyde-dehydrogenase and aldehyde-oxidase (EC 1.2.1.3 and EC 1.2.3.1, respectively), p-450 reductase NADPH cytochrome (EC 1.6.2.4), the demethylase and hydroxylase activities, levels of malonic dialdehyde and the rate of ascorbate-dependent lipid peroxidation, the contents of proteins, phospholipids, cysteine, redox glutathione, glucuronides, sulfates. The level of vitamin A in the animals was found to substantially affect the magnitude of the genotoxic action of NDMA. The supplementary administration of vitamin A reduced the effect of the carcinogen. The mechanism of protective action of retinol was largely explained by the mediated activity of cytochrome-P-450 and glutathione-dependent systems involved in the biotransformation of NDMA. Based on the data available in the literature and their own data, the authors analyzed the effects of retinol on the metabolism of genotoxicants and described possible mechanisms of its antimutagenic and anticarcinogenic action. It is concluded that the effective protection of the body from unfavourable environmental influences may be provided only by supplementary (more than the optimum) intake of vitamin A against the background of a damaging factor.

Effects of pyridoxine deficiency on the metabolism of N-nitrosodimethylamine in the rat.

The alteration in the metabolic activation of N-nitrosodimethylamine (NDMA) was investigated in the rat during dietary pyridoxine deficiency. The in vitro metabolism of NDMA by demethylase system was measured in both liver and kidney microsomes. The profile of the kidney enzyme appears similar to that of the liver indicating that at least two forms of isozymes with the low and the high Km's are present. Pyridoxine deficiency significantly increased the activity of NDMA-demethylase of both organs. The increase in the activity of NDMA-demethylase induced by dietary pyridoxine deficiency can be reversed by supplementation of pyridoxine (500 micrograms), i.p., daily for two consecutive days. The increase in the NADPH cytochrome c reductase activity was observed after 6 weeks on pyridoxine-deficient diet.

[Effects of selenium on endogenous synthesis of N-nitrosamines and toxicity of nitrites in rats]. / Vliianie selena na éndogennyi sintez N-nitrozaminov i toksicheskoe deistvienitritov u krys.

Male Wistar rats were fed selenium (0.2, 2.9 or 5.5 mg/kg of dry feed) in the form of selenomethionine. They also received for 6 weeks amidopyrine (33 mg/kg b.w.) and/or sodium nitrite (20 mg/kg b.w.) administered intragastrically. The quantitation of endogenously synthetized volatile nitrosamines in the stomach, selenium in the serum, biochemically evaluated nitrite and nitrosamines toxicity revealed an inverse relationship between feed selenium concentrations and the amount of endogenously produced nitrosodimethylamine from equal precursor doses. A protective selenium effect on methemoglobin-producing action of nitrite ions was established. It is shown that introduction of precursors induces increased body need in selenium.

Chinese tea inhibits the occurrence of oesophageal tumours induced by N-nitrosomethylbenzylamine and blocks its formation in rats.

Chinese tea can inhibit the occurrence of oesophageal tumours not only by blocking the formation of N-nitrosomethylbenzylamine (NMBzA) in vivo, but also by inhibiting the carcinogenesis of preformed NMBzA. The incidence of oesophageal tumours in rats intubated with preformed NMBzA (5 mg/kg bw per week) and with the precursors of NMBzA (methylbenzylamine, 1 mM/kg bw; nitrite, 0.5 mM/kg bw) without Chinese tea was higher than in tea-treated groups; the same tendency was found with regard to the severity of the oesophageal lesions. The anticarcinogenic effects of five varieties of Chinese tea were different from one another.

[Inhibiting effect of the polyphenolic complex from Plantago major (plantastine) on the carcinogenic effect of endogenously synthesized nitrosodimethylamine]. / Ingibiruiushchee vliianie polifenol'nogo kompleksa iz podorozhnika bol'shogo--plantastina--na kantserogennyi éffekt éndogenno sintezirovannogo nitrozodimetilamina.

Amidopyrine administered in combination with sodium nitrite in the long-term experiment produces the toxic damage of the liver and tumors in rats in connection with endogenic synthesis of carcinogenic nitrosodimethylamine. The inclusion into the animal diet of the polyphenolic complex from Plantago major--plantastine as an inhibitor of the carcinogen synthesis reduced the toxic damage of the liver that was indicated by normalization of biochemical parameters and also decreased the tumor yield from 87.5% to 33.3%. The data obtained may be the basis for the combined use of plantastine with nitrosated drugs that would contribute to carcinogenesis prevention.

Chemoprevention of N-nitrosomethylbenzylamine-induced esophageal cancer in rats by the naturally occurring thioether, diallyl sulfide.

Diallyl sulfide (DAS) is a principal thioether of garlic (Allium sativum) accounting, in part, for the flavor and fragrance of this herb. Previous studies have shown that DAS is a potent inhibitor of experimentally induced colon cancer in mice. Metabolic studies of other garlic-derived substances suggested that DAS could prevent tumorigenicity of other hepatic activated carcinogens. The present study was designed to determine whether DAS could inhibit the DNA-damaging and tumorigenic effects of N-nitrosomethylbenzylamine in rat esophagus. A dose of 200 mg/kg of DAS given p.o. 3 h prior to N-nitrosomethylbenzylamine administration was found to inhibit the carcinogen-induced nuclear toxicity by 64% to 56% at the two doses (3 and 5 mg/kg) of NMBA tested. These results suggested that the compound was potentially anticarcinogenic. In the carcinogenicity experiment it was found that DAS totally inhibited tumor formation in rats treated with a carcinogenic dose of NMBA (100% inhibition of papilloma and squamous cell carcinoma incidence, P less than 0.0001). Additionally DAS was found to substantially reduce hepatic microsomal metabolism of the carcinogen. These data demonstrate that DAS is unique in its anticarcinogenic activity. It strongly suppresses the tumorigenic effects of potent, metabolically activated monoalkylating carcinogens in the gastrointestinal tract.

Effect of dietary selenium on biotransformation and excretion of mutagenic metabolites of N-nitrosodimethylamine and 1,1-dimethylhydrazine in the liver perfusion/cell culture system.

The mutagenicity of N-nitrosodimethylamine (NDMA) and 1,1-dimethylhydrazine (UDMH) has been studied in an isolated liver perfusion/cell culture system. The liver donors, male Wistar rats, were either selenium (Se)-deficient or had a physiologically adequate Se status (Se-supplemented). Mutagenicity was measured in perfusate and bile with Chinese hamster V79 cells as the genetic target. Se deficiency increased the mutagenic effect of NDMA in the perfusate, whereas no mutagenicity was detected in the bile of either Se-deficient or Se-supplemented livers. No significant increase in the mutagenicity of UDMH was seen in the perfusate with Se deficiency, but the bile became mutagenic. Se deficiency thus increased the mutagenicity of both NDMA and UDMH: with NDMA, the effect was observed in the perfusate, and with UDMH, in the bile.

The effects of ascorbic acid deficiency and excess on the metabolism and toxicity of N-nitrosodimethylamine and N-nitrosodiethylamine in the guinea pig.

The influence of ascorbate deficiency and megadosage on the metabolism of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) was investigated in the guinea pig. After 21 days on a scorbutogenic diet, microsomal cytochrome P-450 and cytochrome b5 levels fell by 51 and 32%, respectively, while cytochrome c reductase activity remained constant. The activities of NDMA and NDEA dealkylase I were also depressed significantly. The Vmax of NDMA demethylase I and NDEA deethylase I was significantly depressed. Also, ascorbate deficiency significantly decreased the plasma clearance of both nitrosamines though the LD50 of neither were altered by ascorbate nutrition. Covalent binding of 14C from [14C]NDMA and [14C]NDEA to DNA obtained from liver slices was significantly lower in the deficient than in the control samples; megadosage appeared to have the opposite effect.

Further factors influencing N-nitrosamine formation in bacon.

The possible relationship of unsaturated fatty acids in adipose tissue to the formation of N-nitrosamines in bacon was evaluated by trials in which pigs were fed regular (control), tallow-, coconut fat- and corn oil-supplemented diets. Bacon prepared from pigs fed corn oil-supplemented diets contained significantly higher levels of N-nitrosopyrrolidine and N-nitrosodimethylamine than did control bacon samples; however, bacon produced from pigs fed a coconut fat-supplemented diet contained significantly lower levels of N-nitrosopyrrolidine. Fatty acid analyses of the adipose tissue of the bacon samples indicated that N-nitrosopyrrolidine levels in bacon correlated well with the degree of unsaturation of the adipose tissue. N-nitrosothiazolidine was detected in both brine-cured and dry-cured bacon at levels generally below 4 micrograms/kg. However, its formation was greatly reduced by the inclusion of alpha-tocopherol in the cure. The role of woodsmoke in N-nitrosothiazolidine formation in bacon is discussed.

Influence of dietary thiamin on phenobarbital induction of rat hepatic enzymes responsible for metabolizing drugs and carcinogens.

Male and female Sprague-Dawley rats were fed synthetic diets deficient in or supplemented with thiamin for 2 or 3 weeks. One group of rats receiving the thiamin-supplemented diet was pair-fed the amount consumed by rats fed the thiamin-deficient diet. One-half of each group was administered phenobarbital sodium for four consecutive days prior to decapitation. Rats fed the thiamin-deficient diet had higher NADPH cytochrome c reductase, aniline hydroxylase, and ethylmorphine N-demethylase activities than those fed high levels of thiamin. In addition, these animals generally responded more vigorously to induction by phenobarbital in their synthesis of microsomal protein, and increased activities of NADPH cytochrome c reductase, aniline hydroxylase, and ethylmorphine N-demethylase. Cytochrome P-450 concentration was higher in the microsomes from thiamin-deficient rats and was induced to a greater degree by phenobarbital than in microsomes from rats fed thiamin-supplemented diets ad libitum. Phenobarbital-enhanced metabolism of N-nitrosodimethylamine (DMN) by liver 9,000 g supernatant as evidenced by approximately two-fold increases in formaldehyde formed per gram liver. This increase in DMN metabolism in male rats is due at least in part to the increased concentration of microsomal protein, since metabolism per milligram microsomal protein was not increased. The fact that DMN metabolism per unit of microsomal cytochrome P-450 in phenobarbital-treated animals is decreased to about one-half of that in controls indicates that DMN is either metabolized by a non-cytochrome P-450-dependent system or that it is metabolized by a form of P-450 not induced by phenobarbital. A sex difference was evident in these experiments, females generally being more sensitive to the influence of varying levels of dietary thiamin. Also female rats but not males fed high-thiamin diets responded to phenobarbital with increased DMN metabolism per milligram microsomal protein.

The effects of dietary corn oil on the metabolism and mutagenic activation on N-nitrosodimethylamine (DMN) by hepatic microsomes from male and female rats.

Microsomes from male and female rats fed a diet containing 10% corn oil metabolized N-nitrosodimethylamine (DMN) more rapidly than microsomes from rats fed a similar diet devoid of corn oil. The daily administration of phenobarbital for 4 days prior to harvesting the microsomes resulted in significant induction of the Vmax for N-demethylation of DMN in rats fed the fat-free diet but resulted in no induction (females) or suppression (males) of this enzyme in rats fed the diet containing corn oil. Using concentrations of DMN ranging from 12 to 100 mM, microsomes from rats fed the high fat diet activated DMN to produce mutagenesis in S. typhimurium (TA100) more rapidly than those from rats fed the fat-free diet. Phenobarbital administration induced this activation more effectively in rats fed the corn oil diet than in rats fed the fat-free diet. Phenobarbital induces DMN N-demethylation in rats fed both fat-free and 10% corn oil diets when the DMN concentration is above 10 mM and explains, at least in part, this enhanced mutagenic activation.

A sensitive hepatocyte-mediated assay for the metabolism of nitrosamines to mutagens for mammalian cells.

A sensitive cell-mediated assay has been developed for testing mutagenesis in Chinese hamster V79 cells by carcinogenic nitrosamines. Mutations were characterized by resistance to ouabain and 6-thioguanine. Since V79 cells do not metabolize nitrosamines, mutagenesis in the V79 cells was tested in the presence of primary hepatocytes capable of metabolizing nitrosamines. The hepatocytes were isolated after collagenase and hyaluronidase digestion of liver slices. All seven liver carcinogens of the nine tested nitrosamines exhibited a mutagenic response in this cell-mediated assay. The potent liver carcinogens nitrosodimethylamine, nitrosodiethylamine, nitrosoethylmethylamine, and nitrosodipropylamine could be detected with doses as low as 1 muM. The noncarcinogenic nitrosodiphenylamine was not mutagenic. Nitrosomethoxymethylamine was the only nitrosamine that exhibited mutagenic activity in the absence of hepatocytes, and this activity was diminished in the presence of hepatocytes. It is suggested that the use of hepatocytes prepared by the slicing method for carcinogen metabolism and mutable V79 cells offers a highly sensitive assay for determining the mutagenic potential of carcinogenic nitrosamines and probably of other classes of hazardous chemicals occurring in the environment.

Mediated mutagenesis of dimethylnitrosamine in Neurospora crassa by various metabolic activation systems.

Four metabolic activation systems (growth mediated mycelium extract mediated, host mediated, and organ homogenate mediated) were used to study the mutagenic activity of dimethylnitrosamine (DMN) in both forward and reverse mutation systems in the ad-3 (adenine-3) region of Neurospora crassa. DMN was not mutagenic in Neurospora if conidia alone were treated. It was highly mutagenic, however, if conidia were treated with this compound under any of the four activation systems. Quantitative differences in DMN-induced mutation frequencies were observed between in vivo (growth and host mediated) and in vitro (mycelium extract and organ homogenate mediated) activations. The efficiency of the conversion of DMN to a mutagenic metabolite by the organs of rats and mice appeared to be in a reversed order between the host-mediated (liver greater than kidney greater than lung) and the in vitro organ homogenate-mediated (lung greater than kidney greater than liver) assays. Inductions of reverse mutations in strain N23 indicated that DMN induces base-pair substitution in N. crassa.

Use of human-liver microsomes from kidney-transplant donors for the induction of chromatid aberrations and sister-chromatid exchanges by means of pre-carcinogens in Chinese hamster cells in vitro.

Samples of two human livers taken during operation of kidney donor patients were processed for microsome fractions and used for metabolization of cyclophosphamide (CP) and dimethylnitrosamine (DMN) in combination with the NADPH-generating system. Rat-liver microsomes were checked for comparison. Induction of chromatid aberrations and sister-chromatid exchanges in a newly isolated clone of Chinese hamster fibroblasts served as indicators of activity. Human S-9 fractions standardized on protein content showed strong variations of CP and DMN activation. Whereas liver microsomes of one patient (who also suffered from Gaucher's disease) were highly active for both pre-carcinogens and metabolized DMN at the same level as the uninduced rat-liver microsomes, the S-9 fraction from the second patient failed to activate CP, but was distinctly positive for DMN. It is suggested that samples of liver and other organs of renal transplant donors might be a practicable source of freshly prepared human microsome fractions usable in biochemical, genetic and carcinogenetic studies. Problems concerning the extrapolation of results are discussed.

DNA repair synthesis in primary cultures of kidneys from BALB/c and C3H mice treated with dimethylnitrosamine.

A combined 'in vivo--in vitro' autoradiographic method was employed to examine the DNA repair induced in the kidney by a single dose of dimethylnitrosamine (DMNA). Unscheduled DNA synthesis was found to be dose-dependent in primary kidney cultures of DMNA-treated mice, and practically not detectable in controls. Its amount was positively correlated with the different susceptibility of C3H and BALB/c mice to kidney tumor induction by DMNA. This experimental model appears sensitive and able to provide repeatable results. It may be useful to detect the organotropic activity of a carcinogen toward the kidney.

Interaction of vitamin E and selenium with the hepatotoxic agent dimethylnitrosamine.

Pretreatment of rats with vitamin E, 0.02% w/w of the diet and a sc dose, 200 mg/kg, given 48 hrs. before dimethylnitrosamine, DMNA, was found to ameliorate the acute hepatotoxicity of DMNA (30 mg/kg) as reflected in reduced plasma asparagine-amino-transferase (AspAT) activity. This effect was confirmed by histological evaluation. No significant effect of DMNA on plasma levels of vitamin E was observed, however, DMNA significantly increased the hepatic level of vitamin E supplemented rats. Pretreatment with selenium, 0.5 mg/kg given intraperitoneally 48 hrs. before DMNA, was found to enhance the acute hepatotoxicity of DMNA as reflected in increased elevation of plasma AspAT activity. This effect was not confirmed morphologically. DMNA did not have any effect on the hepatic selenium state in selenium pretreated rats; however, selenium pretreatment tended to decrease hepatic and plasma tocopherol levels. To explain the effects observed in the present investigation, various mechanisms were discussed. If the compounds were acting as antioxidants, then the difference in intracellular localization had to be important. More likely a specific biochemical function involving drug metabolizing enzymes could be involved. Finally vitamin E could protect membranes from damage during the necrotizing action of DMNA.