Mutagenicity of deep-frying fat, and evaluation of urine mutagenicity after consumption of fried potatoes

Mutagen formation during deep-frying was evaluated using standard frying conditions. Portions of pre-fried, sliced potatoes were fried in a commercial brand of hydrogenated vegetable frying fat, which was used repeatedly and for a prolonged period of time. Concentrations of polar oxidation and degradation products, and of dimeric and polymeric triglycerides, were found to increase in the frying fat as well as in fried potatoes with prolonged use of the fat. Thiobarbituric acid-reactive substances were detectable neither in the frying fat nor in the fried potatoes. Polar fractions of repeatedly used frying fat significantly increased the number of revertants in Salmonella typhimurium strain TA97 without S-9 mix. In the presence of S-9 mix mutagenic activity was reduced. As a consequence of ongoing formation of polar degradation and oxidation products, the mutagenicity of the fat increased after repeated use. Polar fractions of lipids extracted from commercially obtained pre-fried potatoes, as well as from fried potatoes, marginally increased the number of revertants in strain TA97 without S-9 mix. The mutagenicity of the lipid fractions of fried potatoes was not related to the heating time of the fat. Methanol extracts of fat-free residues of fried potatoes significantly increased numbers of revertants in strain TA97 after metabolic activation, which indicated that a different class of mutagens had been isolated. The mutagenicity of methanol extracts was not increased after either prolonged or repeated use of the fat. Urine samples of six healthy, non-smoking volunteers, collected during the 24 hr following consumption of portions of potatoes fried in repeatedly used fat, showed no increase in mutagenicity compared with control samples. Since the exact identity of mutagens formed during deep-frying, as well as their metabolic fate in man, is unclear at present, evaluation of possible adverse biological effects associated with consumption of fried foods will require strictly controlled metabolic studies.     

Host-mediated bacterial mutagenesis and enterohepatic circulation of benzidine-derived mutagenic metabolites in rodents

1. Administration of benzidine (100 mg/kg, i.p.) to bile duct-cannulated rats led to a sustained excretion of metabolites in bile which, following glucuronide hydrolysis, were mutagenic to Salmonella typhimurium strain TA98. 2. When the biliary metabolites were re-infused into the duodena of a further group of rats, enterohepatic circulation of mutagens was indicated by extensive re-excretion of biliary mutagens in the recipients. 3. Furthermore, in mouse host-mediated mutagenicity assays, both i.p. injection of benzidine (100 mg/kg) and intracaecal administration of rat biliary metabolites of benzidine produced a mutagenic response in Salmonella typhimurium strain TA98 cells isolated from the liver. 4. The results indicate that enterohepatic circulation adds to the biological persistence of reactive metabolites of benzidine and may contribute to the carcinogenicity of this aromatic amine.     

Safety assessment of heated diacylglycerol oil: Subchronic toxicity study in rats

Diacylglycerol oil is an edible oil with similar taste and usability characteristics as conventional edible oil rich in triacylglycerol oil. The objective of the present study was to evaluate potential adverse effects of heated diacylglycerol and triacylglycerol oil in rats following subchronic administration. The heated diacylglycerol and triacylglycerol oils were prepared separately following deep frying potato slices at 180 degrees C for 8h per day for three days. Sprague Dawley rats were fed diets containing different ratios (concentrations) of heated to unheated diacylglycerol oil. The ratio of heated to unheated diacylglycerol was as follows: 0%/5.5% (control-1; Group 1), 1.0%/4.5% (Group 2), 2.75%/2.75% (Group 3), and 5.5%/0% (Group 4). Two additional groups received the feed containing 5.5% of unheated or 5.5% of heated triacylglycerol oil. Compared to the unheated oils, feeding of heated diacylglycerol or triacylglycerol oil did not reveal any toxicologically significant changes in clinical observation, body weights, body weight gains, feed consumption, ophthalmic examinations, functional observational battery and motor activity, clinical pathology evaluations and organ weights. Similarly, terminal necropsy did not reveal treatment-related gross or histopathology findings. Based on the results of this subchronic study, the no-observed-effect levels (NOELs) of heated diacylglycerol or triacylglycerol oil were 5.5%, the highest levels tested. The mean dietary exposure levels at the highest dose for the heated diacylglycerol and triacylglycerol oil for male and female rats ranged from 3,178 to 4,120 mg/kg/day.     

In vitro antimutagenicity of curcumin against environmental mutagens

The effects of curcumin, the yellow pigment of the spice, turmeric (Curcuma longa) on the mutagenicity of several environmental mutagens were investigated in the Salmonella/microsome test with or without Aroclor 1254-induced rat-liver homogenate (S-9 mix). With Salmonella typhimurium strain TA98 in the presence of S-9 mix, curcumin inhibited the mutagenicity of bidi and cigarette smoke condensates, tobacco and masheri extracts, benzo[a]pyrne and dimethyl benzo[a]anthracene in a dose-dependent manner. Curcumin did not influence the mutagenicity without S-9 mix of sodium azide, monoacetylhydrazine and streptozocin in strain TA100 nor of 4-nitrophenylenediamine in strain TA98. Our observations indicate that curcumin may alter the metabolic activation and detoxification of mutagens.     

Mutagenicity of human urine after the consumption of fried salted salmon

Mutagenicity in the urine of four non-smoking individuals who had eaten salted salmon cooked at home for both lunch and supper was monitored by means of Salmonella/microsome mutagenicity tests. Extracts from fresh and salted salmon had the same level of mutagenicity after being cooked for 10 min at 200 degrees C, but no activity was detected before cooking. Salmonella strains TA98 and TA1538 were equally sensitive to the mutagens and required metabolic activation. No mutagenicity was shown with TA100 and TA1535. Urine samples were tested using a concentrate prepared by means of an XAD-2 resin column. Mutagenicity was detected mainly in urine excreted during 4-5 hr after the ingestion of cooked salmon, but only weak mutagenicity, or none at all, was detected in the urine after the ingestion of vegetables. The levels of urinary mutagenicity due to salmon consumption were not affected when cabbage was eaten simultaneously. The excretion of mutagenic substances was completed within about 20 hr, and there were almost no mutagens in the urine 24 hr after the ingestion of cooked salmon.     

Ames mutagenicity tests of repeatedly heated edible oils

Six cooking oils with different levels of unsaturation were each heated at 180 +/- 3 degrees C for 6 hr on each of four consecutive days and cut potatoes were fried in the oils at hourly intervals. Samples of the heated and unheated oils were tested in the Ames mutagenicity assay. None of the oil samples (taken before or after 24 hr heating) showed any mutagenicity in the Ames test with or without metabolic activation.     

Effect of pyrolysis temperature on the mutagenicity of tobacco smoke condensate.

Tobacco smoke aerosols with fewer mutagens in the particulate fraction may present reduced risk to the smoker. The objective of this study was to test the hypothesis that the temperature at which tobacco is pyrolyzed or combusted can affect the mutagenicity of the particulate fraction of the smoke aerosol. Tobacco smoke aerosol was generated under precisely controlled temperature conditions from 250 to 550 degrees C by heating compressed tobacco tablets in air. The tobacco aerosols generated had a cigarette smoke-like appearance and aroma. The tobacco smoke aerosol was passed through a Cambridge filter pad to collect the particulate fraction, termed the smoke condensate. Although condensates of tobacco smoke and whole cigarette mainstream smoke share many of the same chemical components, there are physical and chemical differences between the two complex mixtures. The condensates from smoke aerosols prepared at different temperatures were assayed in the Ames Salmonella microsome test with metabolic activation by rat liver S9 using tester strains TA98 and TA100. Tobacco smoke condensates were not detectably mutagenic in strain TA98 when the tobacco smoke aerosol was generated at temperatures below 400 degrees C. Above 400 degrees C, condensates were mutagenic in strain TA98. Similarly, condensates prepared from tobacco smoke aerosols generated at temperatures below 475 degrees C were not detectably mutagenic in strain TA100. In contrast, tobacco tablets heated to temperatures of 475 degrees C or greater generated smoke aerosol that was detectably mutagenic as measured in TA100. Therefore, heating and pyrolyzing tobacco at temperatures below those found in tobacco burning cigarettes reduces the mutagenicity of the smoke condensate. Highly mutagenic heterocyclic amines derived from the pyrolysis of tobacco leaf protein may be important contributors to the high temperature production of tobacco smoke Ames Salmonella mutagens. The relevance of these findings regarding cancer risk in humans is difficult to assess because of the lack of a direct correlation between mutagenicity in the Ames Salmonella test and carcinogenicity.     

Mutagens from the cooking of food. II. Survey by Ames/Salmonella test of mutagen formation in the major protein-rich foods of the American diet

The formation of mutagens in the major cooked protein-rich foods in the US diet was studied in the Ames Salmonella typhimurium test. The nine protein-rich foods most commonly eaten in the USA—ground beef, beef steak, eggs, pork chops, fried chicken, pot-roasted beef, ham, roast beef and bacon—were examined for their mutagenicity towards S. typhimurium TA1538 after normal ‘household’ cooking (deep frying, griddle/pan frying, baking/roasting, broiling, stewing, braising or boiling at 100–475°C). Well-done fried ground beef, beef steak, ham, pork chops and bacon showed significant mutagen formation. For chicken and beef steak high-temperature broiling produced the most mutagenicity, followed by baking/roasting and frying. Stewing, braising and deep frying produced little mutagen. Eggs and egg products produced mutagens only after cooking at high temperatures (the yolk to a greater extent than the white). Commercially cooked hamburgers showed a wide range of mutagenic activity. We conclude that mutagen formation following cooking of protein-containing foods is a complex function of food type, cooking time and cooking temperature. It seems clear that all the major protein-rich foods if cooked to a well-done state on the griddle (eggs only at temperatures above 225°C) or by broiling will contain mutagens detectable by the Ames/Salmonella assay. This survey is a step towards determining whether any human health hazard results from cooking protein-rich foods. Further testing in both short- and long-term genotoxicity bioassays and carcinogenesis assays are needed before any human risk extrapolations can be made.     

Effects of human diets on biotransformation enzyme activity and metabolic activation of carcinogens in rat liver

We studied the effects of a complete human diet, based on mean consumption figures in The Netherlands, the heating of food, and the presence of vegetables and fruit in the diet on the drug metabolizing capacity of the rat liver and on metabolic activation of known carcinogens. Groups of five male and five female Wistar rats were given ad lib. one of six different diets for 3 months. Each diet contained 40 energy (E)% fat, 13 E% protein, 47 E% carbohydrate and 5% fibre (w/w). The diets were as follows: a control diet of semi-synthetic materials (A); a human diet of meat, bread and eggs without processing (B); diet B heated under usual household conditions (C); a diet representing a complete human meal including (summer) vegetables and fruit (D); diets consisting of winter vegetables (E) or summer vegetables (F) with fruit. Semi-synthetic components were added to diets B-F to achieve the desired composition. There were differences between male and female rats on the effects of the different diets on hepatic enzyme activity. In female rats, but not in males, ethoxyresorufin-O-deethylase activity was increased significantly (P less than 0.05) in groups C, D and E in comparison with the controls (group A). In male rats ethoxycoumarin-O-deethylase activity was enhanced in groups D, E and F, and glutathione-S-transferase was markedly induced in group F (P less than 0.01). In males, hepatic cytochrome P-450 was significantly (P less than 0.05) increased in groups B, C and E. There was no effect on aminopyrine-N-demethylase activity and almost no effect on UDP-glucuronyltransferase activity in either sex. Microsomes from rats fed heated food (C) markedly increased the mutagenicity of benzo[a]pyrene (B[a]P) in the Ames assay using Salmonella typhimurium strain TA98, in comparison with levels using microsomes from rats fed the raw food (B). Vegetables and fruit decreased B[a]P mutagenicity. All human diets except D decreased the mutagenicity of N-nitrosodimethylamine in tester strain TA100. The results indicate that the influence of components of human diets on rat-liver drug metabolism may have quite different effects on the biotransformation of carcinogens activated by different metabolic pathways.     

Negative Ames tests of epoxide fatty methyl esters derived from hemolysis of linoleic acid hydroperoxides

Five isomeric epoxyhydroxyene and epoxyoxoene fatty esters derived from hemolytic decomposition of linoleic acid hydroperoxide were tested for mutagenicity by the "Ames' top-agar incorporation method using S-9 mix derived from livers of male rats pretreated with Aroclor 1254. The epoxide fatty esters tested--methyl trans-12,13-epoxy-erythro-11-hydroxy-cis(trans)-9-octadecenoate and methyl trans-12,13-epoxy-threo-11-hydroxy-cis(trans)-9-octadecenoate (each composed of approximately 80% cis-9-ene and 20% trans-9-ene), methyl trans-12,13-epoxy-9-oxo-(trans-10-octadecenoate, methyl trans-12,13-epoxy-9-hydroxy-trans-10-octadecenoate and methyl cis-12,13-epoxy-9-oxo-trans-10-octadecenoate--had structural characteristics similar to certain potent mutagens. However, these esters were not mutagenic in Salmonella typhimurium strains TA100, TA98 or TA1537 at concentrations up to 2000 micrograms/test plate. Under the same test conditions, the methyl ester of hydroperoxy linoleic acid, from which these epoxides were derived, was weakly mutagenic in strain TA100 and possibly also in strain TA98.     

Mutagenicity of cysteine and penicillamine and its enantiomeric selectivity

We previously observed that postmitochondrial supernatant (S9) from rat liver and kidney homogenates transforms L-cysteine into a mutagen that reverts bacteria of the strain Salmonella typhimurium TA100 to histidine independence. In the present study the enantiomers of cysteine and penicillamine (beta, beta-dimethylcysteine) have been investigated for mutagenicity. The Salmonella typhimurium strain TA92 was found to be more sensitive than TA100 to the mutagenic action of L-cysteine and was therefore also included. This strain allowed the unambiguous realization of a (weak) mutagenic effect of L-cysteine even in the absence of mammalian enzyme preparations. D-cysteine did not show mutagenicity under any experimental conditions. However, it was strongly bacteriotoxic. On the other hand, both enantiomers of penicillamine exerted clear mutagenic effects. Qualitatively, their mutagenicity was similar to that of L-cysteine in the following respects: the penicillamines were directly mutagenic, their mutagenicity was enhanced by S9, kidney S9 enhanced the mutagenicity more than did liver S9, TA92 was more sensitive than TA100. Thereby it is noteworthy that the ratios of the specific mutagenicities in the two strains were virtually identical in the direct, kidney-S9-mediated and liver-S9-mediated tests suggesting that the ultimate mutagens under these different metabolic conditions were identical. On the other hand, substantial quantitative differences in the mutagenicity between the beta-thiol amino acids were observed. L-penicillamine was about eight times more mutagenic than the clinically used enantiomer, D-penicillamine. In the direct tests, the mutagenic potency of L-cysteine was equal to that of D-penicillamine. In the S9-mediated experiments, the mutagenic potency of L-cysteine was intermediate between those of L- and D-penicillamine.     

Microsomal activation of dibenzo[def,mno]chrysene (anthanthrene), a hexacyclic aromatic hydrocarbon without a bay-region, to mutagenic metabolites

Metabolically formed dihydrodiol epoxides in the bay-region of polycyclic aromatic hydrocarbons are thought to be responsible for the genotoxic properties of these environmental pollutants. The hexacyclic aromatic hydrocarbon dibenzo[def,mno]chrysene (anthanthrene), although lacking this structural feature, was found to exhibit considerable bacterial mutagenicity in histidine-dependent strains TA97, TA98, TA100, and TA104 of S. typhimurium in the range of 18-40 his(+)-revertant colonies/nmol after metabolic activation with the hepatic postmitochondrial fraction of Sprague-Dawley rats treated with Aroclor 1254. This mutagenic effect amounted to 44-84% of the values determined with benzo[a]pyrene under the same conditions. The specific mutagenicity of anthanthrene in strain TA100 obtained with the cell fraction of untreated animals was 6 his(+)-revertant colonies/nmol and increased 2.7-fold after treatment with phenobarbital and 4.5-fold after treatment with 3-methylcholanthrene. To elucidate the metabolic pathways leading to genotoxic metabolites, the microsomal biotransformation of anthanthrene was investigated. A combination of chromatographic, spectroscopic, and biochemical methods allowed the identification of the trans-4,5-dihydrodiol, 4,5-oxide, 4,5-, 1,6-, 3,6-, and 6,12-quinones, and 1- and 3-phenols. Furthermore, two diphenols derived from the 3-phenol, possibly the 3,6 and 3,9 positional isomers, as well as two phenol dihydrodiols were isolated. Three pathways of microsomal biotransformation of anthanthrene could be distinguished: The K-region metabolites are formed via pathway I dominated by monooxygenases of the P450 1B subfamily. On pathway II the polynuclear quinones of anthanthrene are formed. Pathway III is preferentially catalyzed by monooxygenases of the P450 1A subfamily and leads to the mono- and diphenols of anthanthrene. The K-region oxide and the 3-phenol are the only metabolites of anthanthrene with strong intrinsic mutagenicity, qualifying them as ultimate mutagens or their precursors. From the intrinsic mutagenicity of these two metabolites and their metabolic formation, the maximal mutagenic effect was calculated. This demonstrates the dominating role of pathway III in the mutagenicity of anthanthrene under conditions where it exhibits the strongest bacterial mutagenicity.     

Mutagens from the cooking of food. III. Survey by Ames/Salmonella test of mutagen formation in secondary sources of cooked dietary protein

The formation of mutagens in the major cooked protein-rich foods in the US diet was studied in the Ames Salmonella typhimurium test. The nine protein-rich foods most commonly eaten in the USA—ground beef, beef steak, eggs, pork chops, fried chicken, pot-roasted beef, ham, roast beef and bacon—were examined for their mutagenicity towards S. typhimurium TA1538 after normal ‘household’ cooking (deep frying, griddle/pan frying, baking/roasting, broiling, stewing, braising or boiling at 100–475°C). Well-done fried ground beef, beef steak, ham, pork chops and bacon showed significant mutagen formation. For chicken and beef steak high-temperature broiling produced the most mutagenicity, followed by baking/roasting and frying. Stewing, braising and deep frying produced little mutagen. Eggs and egg products produced mutagens only after cooking at high temperatures (the yolk to a greater extent than the white). Commercially cooked hamburgers showed a wide range of mutagenic activity. We conclude that mutagen formation following cooking of protein-containing foods is a complex function of food type, cooking time and cooking temperature. It seems clear that all the major protein-rich foods if cooked to a well-done state on the griddle (eggs only at temperatures above 225°C) or by broiling will contain mutagens detectable by the Ames/Salmonella assay. This survey is a step towards determining whether any human health hazard results from cooking protein-rich foods. Further testing in both short- and long-term genotoxicity bioassays and carcinogenesis assays are needed before any human risk extrapolations can be made.     

Ames mutagenicity tests of overheated brewed coffee

Five kinds of coffee samples were prepared from a commercial drip-grind coffee in order to examine the mutagenicity of brewed coffee using the Ames test. The samples prepared were a thick coffee syrup, coffee solid residues, dichloromethane and ethanol extracts of solid residues, a dichloromethane extract of a distillate from normally heated brewed coffee and dichloromethane extracts of distillates from overheated (150–300°C) brewed coffee. The samples were tested for mutagenicity towards Salmonella typhimurium strains TA98 and TA100 both with and without metabolic activation (S-9 mix). Only the extracts of the distillates obtained from coffee heated to 150° or 300°C exhibited mutagenicity towards strain TA98 with S-9 mix.     

Mutagen excretion and cytochrome P-450-dependent activity in germfree and conventional rats fed a diet containing fried meat

To evaluate a possible role of the intestinal microflora in the metabolism of the highly mutagenic compounds formed in fried meat, conventional and germfree male AGUS rats were fed a semi-synthetic diet containing fried meat. Changes in mutagen excretion in urine and faeces over time were studied using the Ames Salmonella assay. The faecal and urinary extracts were separated by means of high-performance liquid chromatography (HPLC), and the mutagenicity of the collected fractions was determined. Cytochrome P-450 IA (IA1 and/or IA2) were detected by the use of antibodies with the Western blot technique, and the corresponding enzyme activities were measured in microsomes from the small intestine and the liver. A quantitative as well as qualitative difference in excretion of mutagens between germfree and conventional rats was observed. The total excreted of mutagenicity was significantly higher for the conventional than for the germfree rats, as a result of a higher faecal excretion of mutagens in the conventional animals. The HPLC separations of urinary and faecal extracts showed a different mutagenic metabolite pattern between the germfree and conventional rats. An increased activity of the cytochrome P-450-dependent enzyme ethoxyresorufin-O-deethylase was observed in the small intestine of conventional rats on the fried meat diet, whereas no effect of this diet was observed in the germfree rats. Similar results were obtained in immunoblotting experiments using a P-450 IA antiserum. The present study indicates that the excretion pattern and thus also the metabolism of compounds present in fried meat are affected by the germfree status.     

用菌株YG1024对液化石油气燃烧产物接触者尿诱变性的检测

对硝基多环芳烃及其衍生物特异性敏感的菌株已有许多。应用新引进的ＹＧ１０２１和ＹＧ１０２４菌株及传统的“敏感”菌株ＴＡ９８检测了液化石油气燃烧产物接触者尿的致突变性，并对三菌株的敏感性进行了比较。结果显示，在有鼠肝Ｓ９存在时，尿样对菌株ＹＧ１０２４的致突变作用明显高于ＴＡ９８，而相同条件下菌株ＹＧ１０２１的回变数与ＴＡ９８相差不大。提示芳香胺是ＬＰＧ燃烧产物接触者尿中的主要致突变物，菌株ＹＧ１０２４     

Screening of tabacco smoke constituents for mutagenicity using the Ames' test

To clarify the mutagenic activity of individual smoke components, 239 compounds, representative of the gaseous and semivolatile phases of tobacco smoke, were assayed for mutagenicity towards 4 histidine-requiring mutants of Salmonella typhimurium (TA 98, TA 100, TA 1535 and TA 1537). All compounds were tested qualitatively both with and without metabolic activation using a liver fraction (S-9) from Aroclor 1254 or methylcholanthrene induced rats. Without S-9, only 2,3-dimethylindole and 2,3,5-trimethylindole showed mutagenic activity that was not enhanced by hte metabolic activation system. 2,6-Diaminotoluene and coronene, which like the above compounds are not documented carcinogens were found to be mutagenic for strain TA 98 with S-9. Mutagenic activity was also observed for the previously known mutagens benz[a]pyrene, chrysene, benz[a]-anthracene, perylene and β-naphthylamine, on exposure to strains TA 98 and/or TA 100 with S-9.     

Enzyme-mediated mutagenicity in Salmonella typhimurium of contaminants of synthetic indigo products

The mutagenic potential of two natural and seven synthetic, commercial indigo dye products was investigated. The natural products showed no mutagenicity in Salmonella typhimurium stains TA98 and TA100. In the presence of rat-liver homogenate from Aroclor 1254 pretreated rats all of the synthetic products were mutagenic towards strain TA98 but not towards strain TA100. The mutagenic effect produced was highly dependent on the amount of rat-liver homogenate added. Because of its high mutagenic potential, one product was further investigated. In the presence of rat-liver homogenate this product was weakly mutagenic towards strain TA1537 and strongly mutagenic towards strain TA1538. No mutagenicity was observed in strain TA1535. Experiments with purified synthetic indigo and natural indigo revealed that the mutagenic activity of the synthetic commercial products can be ascribed to one or more contaminants.     

沈阳市大气悬浮颗粒物中硝基多环芳烃的观察

采用ＴＡ９８及其缺乏硝基还原酶的衍生菌株ＴＡ９８ＮＲ和ＴＡ９８／１，８ＤＮＰ６，对沈阳市不同功能分区１９９０年第一季度大气悬浮颗粒物进行致突变研究。结果显示，颗粒物的二氯甲烷提取物对上述三菌株均具有明显直接致突变作用。其中对ＴＡ９８ＮＲ和ＴＡ９８／１，８ＤＮＰ６的直接回变数比其母株均降低，ＴＡ９８／１，８ＤＮＰ６的直接回变数降低更明显。提示提取物中含有直接致突变的硝基多环芳烃，其中硝基芘类特别是二硝基芘类在提取物的致突变活性中占重要地位。结果还表明二硝基芘类和１－硝基芘类污染以商业交通区最严重，其次为居民区、工业区。风景区污染相对明显减少。