Safety of the magnetic field generated by a neuronal magnetic stimulator: evaluation of possible mutagenic effects.

OBJECTIVE: The possible mutagenicity of a magnetic stimulus was checked using the Ames test with Salmonella typhimurium TA98 and TA100 as tester strains. METHODS: Samples of these bacteria were exposed to a pulsed magnetic field, on the order of 1 T. The magnetic pulses were generated by a neuronal magnetic stimulator with a flat coil. The magnetic stimulus was a continuous sequence of slightly damped half sinusoids at a rate of 5 pulses/s. Exposure times were 2-5 and 15 min. Exposure position was such as to maximise the magnetic field and minimise the induced electric field. Room temperature was maintained at 28.5 +/- 0.5 degrees C and the temperature was measured inside the samples. RESULTS: None of the exposure conditions showed any increase in mutation in either of the two bacterial strains. CONCLUSIONS: These results are discussed in comparison with effects found in the literature. The magnetic stimulation used under the conditions of this study does not appear to have mutagenic effects. This does not apply to cases where both strong electric and magnetic fields are present.

Prochloraz as potent inhibitor of benzo[a]pyrene metabolism and mutagenic activity in rat liver fractions.

We have determined how prochloraz, an imidazole antifungal agent, affects the metabolism of benzo[a]-pyrene by hepatic microsomes from 3-methylcholanthrene treated male rats. The prochloraz-like 7,8-benzoflavone was a potent inhibitor of total benzo[a]pyrene metabolism while miconazole was a weak inhibitor. The proportion of benzo[a]pyrene dihydrodiols formed was decreased whereas phenols were increased by the in vitro addition of prochloraz. Furthermore, a good correlation was obtained between the effects of prochloraz on the microsomal formation of benzo[a]pyrene metabolites and on the mutagenic activity of benzo[a]pyrene in the Salmonella typhimurium test.

Effect of vitamin A dietary intake on in vitro and in vivo activation of aflatoxin B1.

The mechanism by which vitamin A prevents or delays in chemical carcinogenesis remains unclear. In the present study, we assess the suggestive role of vitamin A in the initiation phase of carcinogenesis. We have conducted a dose-effect relationship between vitamin A dietary intake and aflatoxin B1 (AFB1) genotoxicity measured both in vitro and in vivo. Thus AFB1-induced mutagenesis in Salmonella typhimurium TA98 was investigated and compared to AFB1-induced single-strand breaks (SSBs) in DNA of rat hepatocytes. Rats were fed ad libitum with diet containing 0, 5, 50 or 500 IU of retinyl palmitate for 8 weeks. The AFB1-treated rats were injected i.p. with 1 mg/kg body weight. In the Ames test conditions TA98 back-reversion was negatively correlated with the log of vitamin A concentration in liver S9 fractions from experimental groups. However, the activities of metabolizing enzymes which specifically activate or deactivate AFB1 were found to be significantly decreased in vitamin A-deficient animals and weakly modified in vitamin A-supplemented animals. For in vivo experiments, the DNA elution rate of both AFB1-treated and untreated rats was increased in vitamin A deficiency condition (+79% and +17% respectively) and was reduced with the higher vitamin A dietary level (-44% and -53% respectively). DNA damage measured in vivo showed a significant positive correlation with mutagenic activity measured in the Ames test. These results confirm that the vitamin A status of animals can influence AFB1 genotoxic activity in vitro and indicate that this phenomenon also occurs in vivo. Thus a similar mechanism may be considered for the protective action of vitamin A both in vitro and in vivo. However, this mechanism is unlikely to involve modulation of the microsomal enzyme system responsible for AFB1 metabolism. Therefore a protective mechanism at the cytosolic or nuclear levels may be suggested.

Effects of vitamins A and E on methylazoxymethanol-induced mutagenesis in Salmonella typhimurium strain TA100.

The aim of this study is to report the antimutagenic effect of vitamin A and vitamin E towards methylazoxymethanol (MAM)-induced mutagenesis in Salmonella typhimurium strain TA100 sensitive to alkylating agents. In order to characterize different levels of action of these two fat-soluble vitamins towards the mutagenicity of MAM, several assays have been considered to show the antimutagenic effect and the possible interactions of vitamins with MAM or with the bacteria. Thus, for each vitamin, three different assays with three different incubations have been conducted: (i) MAM, bacteria and vitamins together, (ii) MAM and vitamins, (iii) bacteria and vitamins. The results showed that both vitamins A and E present an antimutagenic effect towards MAM induced mutagenesis. alpha-Tocopherol seems to have an action directly on to the mutagenic agent, whereas the action of retinol is likely due to a protection of the bacterial genoma against MAM. These in vitro results could help to interpret results of colon carcinogenesis studies using animals induced by 1,2-dimethylhydrazine and fed vitamins supplemented diet.

Activation of benzo[a]pyrene and 2-aminoanthracene to bacteria mutagens by mussel digestive gland postmitochondrial fraction.

The ability of the mussel postmitochondrial fraction (S9) to activate benzo[a]pyrene (BaP) and 2-aminoanthracene (2AA) to mutagenic metabolites towards Salmonella typhimurium strain TA98 was tested. The mechanisms involved in this activation were investigated and mussel cytochrome P-450-dependent monooxygenases and its NADPH cytochrome c reductase were found to contribute to the activation of BaP. This activation was improved by treating the mussel with 4,5,4',5'-tetrachlorobiphenyl (TCB) (a 3-methylcholanthrene-type inducer of cytochrome P-450-dependent monooxygenase in marine fish) and was inhibited by alpha-naphthoflavone (ANF), a cytochrome P-450 inhibitor. However, both BaP activation and cytochrome P-450-related metabolic activities are much weaker in mussels than in vertebrates. Mussel S9 activates aromatic amines more effectively than BaP. Pretreatment of mussels with TCB or addition of ANF in the incubation medium has no effect on 2AA activation. As suggested by Kurelec (1985), aromatic amine metabolism may be supported by a flavoprotein mixed-function amine oxidase which is NADPH-dependent.

Effect of vitamin E dietary intake on in vitro activation of aflatoxin B1.

The molecular mechanism of action of vitamin E on mammalian cells remains to be elucidated. In this study, vitamin E dietary intake was assessed for its effects on the initiation phase of carcinogenesis. We have conducted a dose-effect relationship between vitamin E dietary intake and aflatoxin B1 (AFB1) genotoxicity measured in vitro. Thus AFB1 induced mutagenesis in Salmonella typhimurium TA98 was investigated and compared to effect of vitamin E dietary intake on hepatic microsomal P-450 content and specific activities involved in AFB1 metabolism. Rats were fed ad libitum a diet containing 0, 0.05, 0.5 or 5 IU of alpha-tocopherol for 8 weeks. Modulation of vitamin E level in postmitochondrial and microsomal fractions resulted in nutritional effects. Cytochrome P-450 content was not modified by the level of vitamin E in the diet. The microsomal P-450 activities, P-450 IIB1 and IIIA, were decreased in the deficient group to -35% and -16%, respectively, as compared with control diet (0.05 IU). Diet supplemented with 0.5 IU of vitamin E increased P-450 IIB and IIIA activities (+28% and +37%, respectively) whereas a diet highly supplemented in vitamin E (5 IU) reduced these specific P-450 activities. Lipid peroxidation, estimated by the formation of thiobarbituric acid reactive products, increased in the dietary vitamin E free diet (+20%) and strongly decreased in the supplemented group (-99%). This study establishes that in vivo, dietary vitamin E protects directly membrane against damage induced by lipid peroxidation and indirectly hepatic microsomal monooxygenase activities. However, vitamin E accumulation seems to alter membrane structure and function. The nutritional effect of vitamin E on hepatic microsomal cytochrome P-450 activities modified the AFB1 genotoxicity measured in vitro.

Effect of vitamins A, C and glutathione on the mutagenicity of benzo[a]pyrene mediated by S9 from vitamin A-deficient rats.

Vitamin A deficiency has been shown to enhance the mutagenicity of benzo[a]pyrene (Narbonne et al., 1985). Here we report that this is not a result of increased benzo[a]pyrene metabolism but might be a consequence of either a lack of vitamin A or a decreased level of scavengers (ascorbic acid and glutathione) in the liver. However, the addition of vitamin A in vitro in the form of retinyl palmitate strongly inhibits the benzo[a]pyrene mutagenicity. An enhancing effect on the mutagenicity of benzo[a]pyrene is observed with addition of ascorbic acid when incubated with high amounts of the precarcinogen. In vivo addition of high levels of glutathione also reduces the mutagenicity of benzo[a]pyrene.

Lipid peroxidation and benzo[a]pyrene activation to mutagenic metabolites: in vivo influence of vitamins A, E and C and glutathione in both dietary vitamin A sufficiency and deficiency.

Rats fed with either a sufficient-vitamin A or a vitamin A-free diet were pretreated with 750 mg/kg body weight of retinyl palmitate, alpha-tocopherol acetate, ascorbic acid or glutathione. Benzo[a]pyrene (BaP) metabolism and BaP-induced mutagenesis in Salmonella typhimurium TA98 were investigated and related to lipid peroxidation activities in postmitochondrial (S9) liver fraction. The microsomal mixed-function oxidase activities were decreased by vitamin A deficiency and weakly affected by scavenger treatment. The rate of lipid peroxidation of microsomal membranes was unaffected by vitamin A deficiency because of decreased polyunsaturated fatty acids and increased vitamin E contents. However, lipid peroxidation was decreased by pretreatment with fat-soluble vitamins (chiefly vitamin E) and increased by ascorbic acid. Within each experimental group both BaP metabolism and BaP mutagenic activity were closely correlated with the rate of lipid peroxidation. In vitamin A deficiency, the increased BaP metabolism and mutagenicity could be related to a decrease in cytosolic contents of scavengers (vitamin A and glutathione). In Ames test conditions, the free radical pathway became a route for BaP metabolism and thus the BaP activation to mutagenic metabolites is related to the cellular status in free radical scavengers.

Antimutagenicity of components of dairy products.

This paper reports the potential antimutagenicity of some components of dairy products. For both milk and fermented milk, Bifidobacterium sp. Bio (strain Danone 173010), casein and calcium showed a dose-dependent antimutagenic activity against benzo[a]pyrene mutagenicity in the Ames test using Salmonella typhimurium TA98. Fatty compounds present in milk had no antimutagenic effect. At the level occurring in cultured or uninoculated milks, and even more, the bifidobacteria, casein and calcium showed less antimutagenic activity than fermented and uninoculated milks. So, the mix of all these components must contribute to the total protective effect of dairy products against induced mutagenicity, and this does not rule out the possibility of contribution of other unknown substances. The total antimutagenicity of uninoculated skim milk corresponds to the additional activity of casein and calcium. The observed antimutagenic activity of fermented skim milk remains lower than expected.

Structure-activity relationships of the N-methylcarbamate series in Salmonella typhimurium.

Aromatic hydrocarbons of low molecular weight, hydroxy and N-methylcarbamate derivatives were tested for mutagenicity by the reversion of histidine-dependent Salmonella typhimurium TA98 and TA1535 in the presence of a rat-liver 9000 X g supernatant fraction. The presence of 2 or 3 aromatic rings resulted in a weak increase in revertants. Hydroxylation and carbamylation of aromatic rings increased the mutagenic activity of these aromatic compounds. In order to evaluate the structure-activity relationship, the specific molecular connectivity indices were calculated. A significant inverse relationship exists between mutagenicity and zero- and second-order specific molecular connectivity indices. Only compounds with second-order specific molecular connectivity indices lower than 0.300 increased mutagenic activity.

Effects of prototypic PCBs on benzo[a]pyrene mutagenic activity related to vitamin A intake.

The effects of vitamin A dietary intake (2 and 20 IU */g of food) on the mutagenic activity of benzo[a]pyrene (B(a)P) toward Salmonella typhimurium (TA98) were studied either in control rats or in animals treated by the PCB congeners 2,4,5,2',4',5'-hexachlorobiphenyl [2,4,5)2Cl) and 3,4,3',4'-tetrachlorobiphenyl [3,4)2Cl). (3,4)2Cl (a planar compound) strongly increased B(a)P monooxygenase (B(a)PMO) activity and glutathione transferase, (2,4,5)2Cl (a non-planar PCB) was a strong inducer of epoxide hydrolase and a weak inducer of B(a)PMO. Enzyme induction was not modified by changes in vitamin A dietary intake. A higher mutagenic effect was observed in the (3,4)2Cl group than in the (2,4,5)2Cl one. This could be related to the specific form of cytochrome P-450 induced by (3,4)2Cl. In the untreated animals, the activation of B(a)P was higher in the 2-IU group than in the 20-IU one. Conversely, in PCB-treated rats the mutagenic activity of B(a)P was higher in the 20-IU group than in the 2-IU one. PCB induction increased the liver content of vitamin C in both the 2-IU and the 20-IU groups but only increased the glutathione levels in the 2-IU groups. This suggests that glutathione content in cellular fractions may be one of the determining parameters for the mutagenic activity of B(a)P.

Mutagenicity and genotoxicity of sorbic acid-amine reaction products.

Sorbic acid as well as potassium and calcium sorbate (E202 and E203) are legally used as preservatives in numerous processed foods. Owing to its system of conjugated double bonds, sorbic acid is likely to undergo a nucleophilic attack, which may turn it into mutagenic products. The cyclic derivatives resulting from a double addition reaction between sorbic acid and various amines at two different temperatures (50 degrees C and 80 degrees C) have been analysed. A genotoxicity study has been performed with HeLa cells and plasmid DNA. A mutagenesis study has been carried out by using the Ames test. A SOS spot test and a cytotoxicity study have been realised as well. The results showed that the products involved exhibited neither mutagenic nor genotoxic activities.

Genotoxicity study of reaction products of sorbic acid.

Sorbic acid (E200) and its salts (potassium and calcium sorbate: E202 and E203) are allowed for use as preservatives in numerous processed foods. Sorbic acid has a conjugated system of double bonds which makes it susceptible to nucleophilic attack, sometimes giving mutagenic products. Under conditions typical of food processing (50-80 degrees C), we analyzed the cyclic derivatives resulting from a double addition reaction between sorbic acid and various amines. Mutagenesis studies, involving the Ames test and genotoxicity studies with HeLa cells and plasmid DNA, showed that none of the products studied presented either mutagenic or genotoxic activities.

Vitamin A and apoptosis in colonic tumor cells.

The mechanism by which vitamin A prevents or delays chemical carcinogenesis remains unclear. In addition to these antimutagenic and antiproliferative activities, vitamin A seems able to induce programmed cell death. In this study, we assess the suggested role of vitamin A on the in vitro apoptosis induction in a rat colonic tumor cell line. Several concentrations of retinyl palmitate were added in the culture media. We observed cell proliferation by measuring the (3H)thymidine incorporation, cell differentiation by measuring the intestinal alkaline phosphatase expression, and apoptosis induction by DNA fragmentation and morphological evolution of adherent and floating cells. The results show that vitamin A decreases (3H)thymidine incorporation after 1 day of treatment, induces alkaline phosphatase expression, and increases the number of cells falling in apoptosis. This report confirms the role of vitamin A on the induction of cell differentiation, on the inhibition of cell proliferation and shows the vitamin A capacity to induce apoptosis. These results could be attractive to prevent development of colon cancer by vitamin A supplemented diets.

Lack of aberrant crypt promotion and of mutagenicity in extracts of cooked casein, a colon cancer-promoting food.

Dietary casein cooked at 180 degrees C promotes the growth of aberrant crypt foci and colon cancer in rats initiated with azoxymethane. We speculated that promotion was due to a product that could be extracted by a solvent, such as 5-hydroxymethyl-2-furaldehyde (HMF), with tumor- promoting activity or the carcinogenic heterocyclic aromatic amines (HAA). This hypothesis was tested by extracting cooked casein with solvents and water. The extracts were then 1) assayed by high-performance liquid chromatography for HMF and HAA, 2) measured for mutagenicity on a frame-shift-sensitive strain of Salmonella typhimurium, and 3) fed for 100 days to azoxymethane-initiated rats to test the promoting effect on aberrant crypt foci. Data show that 1) no HMF or HAA was detected in cooked casein, 2) no mutagenicity was detected on strain TA98, with or without metabolic activation, and 3) promotion was not associated with the extracts but with the cooked casein residue. Therefore the promotion by cooked casein would not appear to be associated with a product that can be extracted by solvents.

Mutagenicity and genotoxicity of sorbic acid-amine reaction products.

Sorbic acid (E200) and its salts (potassium and calcium sorbate: E202 and E203) are allowed for use as preservatives in numerous processed foods. Sorbic acid had a conjugated system of double bonds which makes it susceptible to nucleophilic attack, sometimes giving mutagenic products. Under conditions typical of food processing (50-80 degrees C), we analysed the cyclic derivatives resulting from a double addition reaction between sorbic acid and various amines. Mutagenesis studies, involving Ames' test and genotoxicity studies with HeLa cells and plasmid DNA, showed that none of the products studied presented either mutagenic or genotoxic activities.

Effects of resistant starch- and vitamin A-supplemented diets on the promotion of precursor lesions of colon cancer in rats.

We have evaluated the potential protective effect of resistant starch (RS)- and vitamin A-supplemented diets on the promotion of preneoplasic lesions of rat colon, aberrant crypt foci (ACF), induced by 1,2-dimethylhydrazine dihydrochloride (DMH). We have tried to show whether the association of these two dietary constituents in the same diet could have synergistic effects. RS, vitamin A, and RS+ vitamin A were incorporated into the rat diets. Experimental diets were given one week after DMH injection and maintained for 12 weeks until the animals were sacrificed. The total number of ACF decreased with the three experimental diets. For RS- and RS + vitamin A-supplemented diets, this decrease is primarily due to a decrease in small ACF. For the vitamin A-supplemented diet, small and large ACF have a tendency to decrease. The effects of the diets on parameters influencing colon carcinogenesis were also studied. Only RS- and RS + vitamin A-supplemented diets have modified cecal pH, fecal and cecal butyrate contents, fecal excretion, cecal weight, and colon length. Vitamin A has been observed in colonic epithelial cells of rats receiving vitamin A- and RS+ vitamin A-supplemented diets. The association between RS and vitamin A shows neither a cumulative nor a synergistic protective effect.

Effect of dairy products on initiation of precursor lesions of colon cancer in rats.

This study reports the modulating effect of some dairy products on initiation of putative preneoplasic lesions in rat colon (aberrant crypts) by 1,2-dimethylhydrazine dihydrochloride. Uninoculated skim milk, skim milk fermented with Bifidobacterium sp Bio (Danone strain 173010), and a suspension of the same lactic acid bacteria were incorporated in the animals' diet. The tested diets significantly reduced the incidence of aberrant crypts compared with the control diet by 51%, 49%, and 61%, respectively. The effects of the diets on cecal pH, hepatic UDP-glucuronyltransferase activity, and cecal microflora enzyme beta-glucuronidase were also studied. There was no significant difference in cecal pH between rats fed experimental diets and control rat. The diet supplemented with the Bifidobacterium strain suspension significantly decreased only the cecal beta-glucuronidase activity. Both enzyme activities were reduced in rats fed fermented skim milk- or uninoculated skim milk-supplemented diets compared with control animals.

Hyperlipidic diets induce early alterations of the vitamin A signalling pathway in rat colonic mucosa.

OBJECTIVE: Dietary factors can be associated with colorectal cancer. Fatty acids modulate gene expression in various tissues, mediated by activation of the peroxisome proliferator activated receptor: PPAR. Vitamin A signalling is mediated by retinoic acid (RA) receptors (RAR) and retinoid X receptors (RXR). The steroid nuclear receptors PPAR, RAR, RXR, are DNA-binding proteins and they induce gene transcription upon activation by specific ligands and interacting with distinct promoter sequences in the target genes. The aim of this study was to investigate the impact of hyperlipidic diets on the expression of PPARg, RXRa and RARb mRNA in rat colon. METHODS: Rats were fed during 4 weeks with the following diets: a cafeteria diet where 60% of the energy was supplied as lipids and a high fat diet (HFD) represented by 25% of a safflower oil (w/w) rich in polyunsaturated fatty acids, mainly n-6. Nuclear receptors mRNA were quantified by realtime RT-PCR with TaqMan probe process or SYBRGreen I chemical. RESULTS: The cafeteria diet and the HFD induced a significant decrease in RARb mRNA: -36% (p<0.02) and -64% (P<0.001) respectively. Simultaneously, an increased expression of PPARg mRNA was observed for cafeteria diet +35% (P<0.05) and for HFD +45% (P<0.05). The level of RXRa mRNA was significantly increased for cafeteria diet: +53% (P<0.0002), while no significant difference in RXRa mRNA was observed in colonic mucosa rats whose fed with the 25% HFD. CONCLUSIONS: These results showed that an hyperlipidic diet could induce early modifications in the pattern of expression of nuclear receptors in rat colon. Many mechanisms could be probably involved but one hypothesis is that a modification of the balance between the nuclear receptors, resulting from an increased expression of PPARg, could induce a decreased expression of RARb in rat colon.

Effect of resistant starch and/or fat-soluble vitamins A and E on the initiation stage of aberrant crypts in rat colon.

This study reports the modulating effects of resistant starch (RS) and the fat-soluble vitamins A or E, alone or in combination, on initiation of preneoplastic lesions in rat colon aberrant crypt foci (ACF) induced by 1,2-dimethylhy-drazine. One group of male Sprague-Dawley rats was fed a basic diet and five groups were fed experimental diets supplemented with 25% RS, 200 IU vitamin A, 5 IU vitamin E, 25% RS + 200 IU vitamin A, or 25% RS + 5 IU vitamin E for four weeks. After induction by 1,2-dimethylhydrazine, all the animals were fed basic diets for four more weeks before sacrifice. Compared with the basic diet, only the vitamin A-supplemented diet significantly reduced the incidence of ACF. The vitamins incorporated in the animals' diets increased the vitamin concentrations in hepatic and colonic cells compared with the animals fed the basic diet. The preventive effect of vitamin A seems to be due to a direct effect on colonic epithelial cells. The three diets supplemented with RS significantly decreased cecal pH and bacterial beta-glucuronidase activity and increased cecal weight and fecal output. The retrograde high-amylose maize, type 3, used in this study does not significantly decrease ACF. This RS has an effect on the colon similar to that of nonstarch polysaccharides. Neither biochemistry nor four weeks of dietary supplementation is likely sufficient for adaptation of the rat colonic flora.